Cross-protective pathogen protection, methods and compositions thereof

ABSTRACT

The present disclosure provides a method of inducing a cross-protective immune response in a subject against a pathogen, such as influenza, comprising administering a first unique pathogen antigen to the subject; and administering a second unique pathogen antigen 3-52 weeks after a); wherein the second unique pathogen antigen and the first unique pathogen antigen are immunologically distinct but share conserved sites that are not normally immunogenic for antibodies. Also disclosed herein are assays for detecting cross-protective antibodies, methods of generating novel cross-protective antibodies. Further provided are novel antibodies against influenza.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry of PCT/CA2011/000817, filedJul. 22, 2011 (which designates the U.S.), which claims priority fromU.S. Provisional Patent Application No. 61/366,747 filed Jul. 22, 2010,which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“14636-P37132US01_SequenceListing.txt” (32,768 bytes), submitted viaEFS-WEB and created on Jan. 21, 2013, is herein incorporated byreference.

FIELD OF THE DISCLOSURE

The disclosure relates to cross-protective and novel immunologicalcompositions and vaccines for pathogen protection, in particular, viralprotection such as influenza protection. The disclosure also relates tomethods of inducing an immune response that protects against a broadspectrum of pathogenic strains and subtypes and methods of vaccinationthereof.

BACKGROUND OF THE DISCLOSURE

Protruding from the membrane of the influenza A viruses are twoproteins, the hemagglutinin (HA) and the neuraminidase (N). Thehemagglutinins are subject to extreme variation through mutation andselection by the antibodies of the host animal. The hemagglutinins ofinfluenza A viruses circulating in different animals fall into one of 16evolutionarily related families called subtypes (called H1, H2 . . .H16). The hemagglutinins are further divided according to phylogenybetween two related groups, Group 1 (H1, H2, H5, H6, H8, H9, H11, H12,H13 and H16) and Group 2 (H3, H4, H7, H10, H14 and H15). Theneuraminidase likewise falls into 9 families. Influenza A viruses areclassified by the subtype of hemagglutinin (HA) and the neuraminidase(N) they exhibit. The influenza A viruses currently circulating inhumans (H1N1 and H3N2) are respectively of the H1 and H3 subtypes ofhemagglutinin and N1 and N2 subtypes of neuraminidase. The protectiveimmune response to seasonal influenza viruses is dominated byisolate-specific, subtype-specific, neutralizing antibodies that bindstrongly to the head of the HA, thereby blocking the function of the HAproteins in attaching the virus to the host-cell receptors (Wiley et al.1987). “Antigenic drift”, the selection of strains of viruses withmutations on the surface of the HA head that decrease binding ofneutralizing antibodies so that they do not protect against the newmutated (“drifted”) strain of virus, creates the regular need forupdated seasonal influenza vaccines. The HA head of the novel 2009(H1N1) pandemic influenza A virus of swine origin (nH1N1) wasantigenically distinct (“antigenic shift”) from H1N1 seasonal influenzaviruses that had been circulating in humans (Xu et al. 2010) and thusmost humans lacked protective antibodies (Itoh et al. 2009).

The hemagglutinin protein mediates infectivity of the influenza virus,first binding the virion to the host cells, and second, fusing themembrane of the virus to the host cell membrane, enabling the viralgenome to enter the cells (Wiley et al. 1987). The hemagglutinin proteinhas a globular head and a stem: the head of the hemagglutinin mediatesthe attachment of the virus to the host cells, whereas the stem of thehemagglutinin mediates the fusion of the membrane of the virus to thehost cell membrane. Antibodies against the head of the hemagglutininthat only bind to the hemagglutinin of one strain or isolate ofinfluenza virus (isolate/strain-specific) predominate in the usual humanimmune response to the seasonal influenza. If antibodies against thehead of the hemagglutinin are of sufficient affinity/avidity and theysterically inhibit the receptor-binding site, they block infectivity ofthat strain/isolate of a sub-type of virus by inhibiting binding of thevirus to the host cell.

Antibodies to the head of the hemagglutinin give protection from beinginfected twice by the same strain of influenza virus but theseprotective antibodies are very specific for a given isolate/strain of asubtype of influenza virus and thus only neutralize and protect againstspecific isolate/strains of influenza viruses (Wiley et al. 1987). Asthe replication of the influenza virus genome is very prone to errors,mutants of the virus readily arise. Those mutants that escapeneutralization by antibodies against the head of the hemagglutinin ofthe original strain of virus tend to be selected to replicate becausethe new, mutant hemagglutinin has so low affinity for the protectiveantibodies against the original isolate/strain, that the antibodies canno longer neutralize the mutant virus. The mutant virus can thenre-infect people immune to the original influenza virus giving rise to anew isolate of seasonal influenza. This process is called “antigenicdrift” and explains why a new seasonal influenza vaccine, made up of themost dominant mutant strains of the circulating strains of influenzavirus to induce neutralizing antibodies to the new mutant virus, isneeded recurrently. Many mutations in the head of the hemagglutininaround the receptor-binding site that disrupt binding of neutralizingantibodies to the original isolate/strain of the virus arewell-tolerated by the virus because they do not interfere with receptorbinding.

There is another, more drastic change in the antigenicity of thehemagglutinin termed “antigenic shift”, that is characteristic of theviruses that cause influenza pandemics. The Spanish H1N1 influenza 1918pandemic is estimated to have killed 50 million humans. Many subtypes ofinfluenza virus circulate in animals, mainly aquatic birds, andinfection of humans with these animal viruses can cause serious humaninfections, with highly pathogenic avian H5N1 influenza having amortality of over 50% (Yen and Webster, 2009). Antigenic shift can occurwhen different strains of influenza, circulating in birds, swine andhumans, infect the same host, enabling reassortment of genetic material,which is present on 8 pieces of RNA. Alternatively it can occur whenviruses that are circulating in another species other than humans,infect humans and replicate in humans and transmit between humans. Inthe case of the 1918 “Spanish” H1N1 influenza pandemic, the influenzavirus may have obtained all its 8 gene segments from avian species (Yenand Webster, 2009). In the case of the 1957 H2N2 influenza pandemic andthe 1963 H3N2 influenza pandemic, reassortment between human influenzastrains and H2 or H3 from avian species was involved (Yen and Webster,2009). The 2009 pandemic influenza A H1N1 virus (pdmH1N1) was generatedby reassortment between two swine influenza viruses, each with genesfrom avian, swine and human influenza viruses (Ding et al, 2009, Gartenet al, 2009). Thus the HA of the 1918 H1N1, 1957 H2N2, 1968 H3N2 and2009 pandemic influenza viruses ultimately originated from avianinfluenza. The HA of pdmH1N1 is distantly related to the 1918 pandemicinfluenza H1N1 virus (Xu et al, 2010). The 2009 pandemic influenza AH1N1 virus (pdmH1N1) was generated by reassortment between two swineinfluenza viruses, each with genes from avian, swine and human influenzaviruses (Ding et al, 2009, Garten et al, 2009). The next pandemic mayhave genetic contributions from the highly pathogenic H5N1 avianinfluenza (H5N1) (Yen and Webster, 2009). Moreover, if the highlypathogenic H5N1 avian influenza (H5N1) undergoes genetic changes thatmake it readily transmissible in humans, it may become a pandemic.

The differences between the amino acid sequences of the ectodomain ofthe hemagglutinin of pandemic influenza viruses and the currentcirculating seasonal influenza viruses are substantial (“antigenicshift”). For example in the hemagglutinin of the 2009 pandemic H1N1influenza, 21% of the amino-acid sequence of the ectodomain wasnon-identical with the corresponding sequence in seasonal H1N1 virus and˜50% in the key epitopes on the HA head were non-identical (Xu et al,2010). In addition, mutations in the hemagglutinin of human seasonalH1N1 influenza viruses (but not the HA of the 2009 pandemic H1N1influenza virus that was derived from a swine H1N1 influenza virus) hadintroduced glycosylation sites in the head, blocking the access ofneutralizing antibodies to the hemagglutinin head (Wei et al 2010a). The2009 H1N1 virus spread so quickly it became a pandemic because the humanpopulation at that time, especially young people, had no circulatingprotective antibodies that could neutralize it (Itoh et al, 2009).Although there can be up to 20% amino acid differences in hemagglutininswithin subtypes there can be 30-70% differences between the sequences ofdifferent subtypes of hemagglutinins (Karlsson Hedestam et al 2008). Forexample, the ectodomain of the hemagglutinin of an isolate of highlypathogenic avian influenza H5N1 exhibits ˜36% non-identical amino acidsin the corresponding sequence in seasonal H1N1 influenza or the pandemic2009 H1N1 influenza virus.

In contrast to mutations in the head of the hemagglutinin, mutations inthe stem region of hemagglutinin are not well-tolerated because mostmutations in the stem disrupt its structurally constrained role inmediating the fusion of the viral membrane to the host cell membrane,which is essential for infectivity. Thus different isolates and evensubtypes of influenza virus exhibit little variation in the regions ofthe hemagglutinin stem that control fusion (Sui et al, 2009). Rareantibodies that bind to the hemagglutinin stem can neutralize influenzaviral infectivity by inhibiting the conformational change in the stalkand thus the fusion of the viral membrane and the host cells membrane(Throsby et al, 2008, Sui et al, 2009, Ekiert et al, 2009). Because thestalk is conserved over many subtypes of influenza virus, the“heterosubtypic” antibodies that target the conserved sites of thehemagglutinin stem can neutralize multiple isolates and subtypes ofinfluenza virus (Throsby et al, 2008, Sui et al, 2009, Ekiert et al,2009).

However, for reasons that are not understood by those skilled in theart, antibodies against the hemagglutinin stem that can bind to thehemagglutinin stem of many isolates/strains and subtypes of influenzaviruses (“cross-protective or “heterosubtypic” antibodies) are notinduced at high frequency in normal infections or vaccinations withseasonal influenza. Corti et al. 2010) reported that heterosubtypicmemory B cells were undetectable in normal humans but after seasonalinfluenza vaccination they could be detected in some individuals,although the frequency after seasonal influenza vaccination was very lowand variable. They generated monoclonal antibodies from these rareheterosubtypic memory B cells and all but one bound to the hemagglutininstem. However, the frequency of the heterosubtypic memory B cells afterseasonal influenza vaccination was 26-200 fold less than that of memoryB cells making antibodies specific for the seasonal influenza vaccine.The question of whether these heterosubtypic memory B cells actuallygave rise to plasmablasts that secreted antibodies in response toseasonal influenza vaccine was not addressed in Corti et al (2010).Corti et al. (2010) did report that they detected, using a verysensitive assay, a small amount of heterosubtypic antibody in the serumthat was insufficient to neutralize the H5N1 influenza virus. Corti etal. (2010) acknowledged that the magnitude of the response they saw wasnot useful for protection and finished their paper with “even in highresponder individuals, heterosubtypic antibodies hardly reach effectiveneutralizing concentration in the serum.” Wrammert et al (2011) reportedthat they had generated monoclonal antibodies from newly generated bloodplasmablasts shortly after seasonal influenza vaccination and found thatnone of the monoclonal antibodies were heterosubtypic antibodiestargeted to the hemagglutinin stem.

The extremely low levels of cross-protective antibodies that bind withhigh affinity to the hemagglutinin of different isolates/strains andsubtypes of virus (“cross-reactive or “heterosubtypic” antibodies)induced by infection (Wiley at al 1987) or vaccination (Corti et al,2010) with seasonal influenza, explains why infection or vaccinationwith a given strain of seasonal influenza virus does not protect againstother isolates/strains or subtypes of influenza virus. The lack ofcross-protective and heterosubtypic antibodies is surprising given thatmost humans have been infected or vaccinated multiple times withdifferent isolates/subtypes, with at least two subtypes of seasonalinfluenza (H1N1 and H3N2) and in some older individuals, also with theH2N2 virus.

Artificially engineered antibodies have been generated against theconserved region of the HA stem and they have been shown to neutralizemultiple strains and subtypes of influenza (Throsby et al. 2008, Sui etal, 2009). They were generated by shuffling a library of humanimmunoglobulin heavy-chain and light-chain genes expressed inbacteriophages and selecting the resultant antibody fragments that boundto the H5 hemagglutinin of avian influenza (H5N1). These antibodiesbound not only to H5 hemagglutinin but also to hemagglutinins fromnumerous other influenza subtypes, with the notable exception of H3 andH7 hemagglutinins from Group 2. Most of these artificial antibodies usedone IGHV1-69 gene, and two studies showed by X-ray crystallography thatthe CDR1 and CDR2 encoded by this germline gene made the key contacts bythese antibodies with the stem region of the H5 hemagglutinin (Ekiert etal. 2009; Sui et al. 2009). The light chain made minimal or no contactswith the hemagglutinin. This gene IGHV1-69 is expressed by most humansand therefore these heterosubtypic antibodies using IGHV1-69 would beexpected to be made by most humans in large quantities given therecurrent antigenic stimulation with infections or vaccinations withseasonal H1N1 influenza. One group looked at the donor of theimmunoglobulin gene library that yielded these artificially generatedcross-reactive antibodies, and found that the donor did not have anycirculating cross-reactive antibodies. Moreover Sui et al. (2009)concluded that such antibodies were not found amongst a large number ofanti-influenza monoclonal antibodies cloned out of donors who had beenvaccinated against seasonal influenza (Wrammert et al. 2008), and thatsome mechanism in the human immune system prevented these cross-reactiveantibodies against the stem of hemagglutinin being produced in humans.There has been considerable interest in designing vaccines based onthese observations (Chen et al. 2009). Corti et al. 2010 speculated thata new vaccine with an engineered immunogen that better exposed the stemregion of the HA to achieve optimal presentation to B cells might resultin heterosubtypic, cross-protective antibody responses. These attemptsat producing a broad spectrum influenza vaccine have involvedconstructing artificial versions of the stem region of the hemagglutinin(Sagawa et al. 1996 and Steel et al. 2010) and have been based on thethesis that the stem of the hemagglutinin was masked by the bulky headdomain, which was thus immunodominant (Steel et al. 2010; Wang et al2010). However, although there was some protection induced byimmunization with the “headless” HA, these researchers found no evidence(Sagawa et al. 1996) or very marginal evidence (Steel et al. 2010) thatthe protection was due to heterosubtypic neutralizing antibodies thatcould be transferred by serum from vaccinated mice. Another approachtried recently was to alter the presentation of the hemagglutinin byusing DNA vaccination followed by a protein or viral vector boost (Weiet al, 2010b) which induced antibodies against the HA stem. However itwas not shown by transfer of serum from vaccinated animals that theantibodies provided protection. These experiments were only done innaïve animals that had no previous experience of influenza vaccinationor infection. The authors acknowledged that they might find differentresults, with human populations that had been previously exposed toinfluenza hemagglutinin.

Karlsson Hedestam et al (2008) and Kwong and Wilson (2009) drew aparallel with influenza virus and HIV-1 with respect that both virusesare extremely variable and both elicit isolate/strain-specificneutralizing antibodies against epitopes on peptide loops that bothviruses can readily mutate and thus escape from the neutralizingantibodies induced by the original strain. In both diseases raremonoclonal antibodies have been generated that can neutralize a broadrange of isolates and strains of viruses. Karlsson Hedestam et al (2008)and Kwong and Wilson (2009) pose the challenge to induce these broadlyneutralizing antibodies by vaccination and contemplate new immunogens.

Antibodies are proteins that circulate in the bloodstream and have theability to bind to and neutralize viruses and toxins and otherpathogens. Antibodies come in billions of configurations and, given thisstructural diversity, it is likely that one or more of the antibodies inan individual will bind to any foreign substance or virus. The cells ofthe blood and immune system that make antibodies are termed “B cells”.Each B cell makes only one of the billions of different types ofantibodies and has samples of that antibody displayed on its surface.When foreign substances (termed “antigens”), like the influenza virus,enter the body, they bind to those rare B cells that make a specificantibody that binds that foreign substance and stimulate those B cellsto multiply. The multiplying B cells then undergo a process called“affinity maturation” in a structure called a germinal centre thatdevelops in a lymph node. In this affinity maturation process, the genesencoding antibodies in the B cells accumulate somatic mutations. In thegerminal centre, those B cells that make a mutated antibody that bindstightly to the antigen are selected. To undergo this “affinitymaturation” process, B cells need the help of a related cell in theimmune system called the “T cell” and the process of selection of the Bcells that make the highest affinity antibodies is intimately involvedwith the mutual interactions between B cells and T cells (Allen et al,2007, Victora, et al 2010, Schwickert et al 2011). Those B cells thatmake antibodies that bind to the hemagglutinin of an influenza virus canbind to the hemagglutinin or more likely to the virion and theninternalize the protein or the virus (Russell et al, 1979). The B cellsdigest the hemagglutinin or virus and present small parts of theproteins (peptides) to the helper T cells. B cells need to present thepeptide antigen to T cells in order to form a tight conjugate that willensure that the T cell stimulates the B cell to proliferate and enterthe germinal centre (Schwickert et al 2011) and then to proliferate andsurvive in the germinal centre (Allen et al, 2007, Victora, et al 2010).

The mechanism of this “affinity maturation” process involves two steps,first the induction of mutations in the antibody genes in B cells, andsecond, the survival of those B cells that make a more tightly binding(“higher-affinity”) antibody. The advantage that B cells that make therelatively higher affinity antibodies have over B cells that make lesseraffinity antibodies results from them being better able to presentpeptide antigens to T helper cells. There is a limiting number of Tcells that have receptors specific for the hemagglutinin or the proteinsin the influenza virion and they form conjugates preferably with the Bcells that make relatively higher affinity antibodies. Theseantigen-specific T cells become activated and stay in contact with the Bcells while they induce them to proliferate and enter the germinalcentres. When the B cells have entered the germinal centre, they need topresent peptide antigens to the helper T cells to proliferate andsurvive. It has been recently established by elegant experiments thatthe relative affinity/avidity of the antibody expressed by a B celldetermines its relative ability to compete with other B cells forpresenting antigen to T helper cells (Victora et al 2010, Schwickert etal 2011). Thus B cells with higher affinity for the antigen willmonopolize the T cell help.

The B cells that make the highest affinity antibodies and that thussurvive the process of affinity maturation, become two types ofspecialized cells that leave the germinal centre and enter the blood,one specialized to secrete large amounts of antibodies(“antibody-secreting cells” or “plasma cells”), and one specialized tocirculate in the blood for very long durations termed a “memory B cell”.If the memory B cell re-encounters the same foreign substance or antigenthat induced it, it responds by rapidly producing antibodies specific tothe antigen, making the individual “immune” to that antigen.

Memory B cells can live for decades in the body. Indeed circulatingantibodies persisted in elderly humans for over 90 years after the 1918pandemic. Similarly, neutralizing high-affinity monoclonal antibodieshave been cloned from memory B cells binding to the head of thehemagglutinin of the 1918 pandemic H1N1 influenza virus from elderlyhumans, from people who were born before the pandemic meaning that thememory B cells had persisted for over 90 years (Yu et al, 2008). Thisexemplifies the longstanding, selective pressure that human influenzaviruses are under by high-affinity, neutralizing antibodies against thehemagglutinin head. Moreover it indicates that memory B cells againstthe hemagglutinin head are a constant feature of the human immune systemand their presence and specificity and affinity must be taken intoaccount if it is contemplated to undertake influenza vaccination. Ifmemory B cells re-encounter their specific antigen they can respondquickly in two ways. If they bind tightly to the antigen (ie theantibody the memory B cell makes is of high-affinity), they transforminto a plasmablast, a cell specialized for secreting large amounts oftheir specific antibody (Paus et al 2006). If they bind more weakly toan antigen, for example a mutated version of the original antigen likean escape mutant of hemagglutinin from an “antigen-drifted” influenzavirus, they re-enter the affinity-maturation process and undergoselection to bind more tightly to the new antigen (Paus et al 2006).Memory B cells that make antibodies that bind relatively weakly to thehemagglutinin of an influenza virus can still bind to the hemagglutininor to the virion and ingest the protein or the viral proteins(Schwickert et al, 2011). Thus memory B cells that make antibodies thatneutralize an original isolate of seasonal influenza but that do notneutralize a “drifted” isolate of seasonal influenza, can still readilybind hemagglutinin and present peptides from the hemagglutinin or aninfluenza virion to helper T cells. There are many shared T cellepitopes between different isolates and subtypes of influenza viruses(Doherty et al, 2008). Moreover T cell epitopes can come from all partsof the primary sequence of the protein, even the internal parts of theprotein that are not displayed on the surface. Moreover if a B cell ismaking an antibody that binds to hemagglutinin, that B cell can bind andinternalize a virion or a fragment of it. That B cell can then presentto a T cell and get help from many T cells specific for individualpeptides from the internal, conserved proteins of the virion (Russell etal, 1979).

SUMMARY OF THE DISCLOSURE

The present inventor has provided evidence that serial immunization ofhumans previously exposed to influenza infections or vaccinations withcompositions comprising antigenically distinct and unique hemagglutininmolecules, encompassing the whole or part of the ectodomain, providesfor the dominance of a minor subset of B cells that make heterosubtypicor cross-reactive antibodies that bind regions of the HA that areconserved or shared between different isolates/strains and subtypes ofinfluenza A virus. The fact that the subject to be vaccinated has notpreviously encountered the unique, distinct antigenic nature of thesetwo (or more) specified hemagglutinins is critical as it avoids theactivation of dominant memory B cells induced by previous seasonalinfluenza vaccinations or infections. The fact that there is a very lowfrequency of memory B cells to the unique hemagglutinin in thepopulation to be vaccinated (for example, undetectable or less than 1per million, or at least less than 0.01% of IgG-expressing memory Bcells circulating in the blood making antibodies reacting strongly tothe unique antigen) can be readily ascertained using the methods of Wenet al 1987 or Corti et al 2010. The disclosure thus provides a means toavoid activation of the memory B cells which, in a normal vaccination orinfection with seasonal influenza would successfully outcompete for Tcell help that minor subset of B cells that make cross-reactive,cross-protective antibodies that bind to conserved regions of the HA.Because T cells recognise T-cell epitopes shared between viral subtypes(Doherty et al 2008), the use of unique hemagglutinins or inactivatedviruses or attenuated influenza viruses ensures that there is ample Tcell help for the B cells making heterosubtypic, cross-protectiveantibodies, in contrast with using designed immunogens comprising thehemagglutinin stem (Sagawa et al. 1996, Steel et al. 2010).

Accordingly, the present disclosure provides a method of inducing across-protective antibody response in a subject against a pathogencomprising:

(a) administering a first unique pathogen antigen to the subject;

(b) administering a second unique pathogen antigen 3-52 weeks after a);

wherein the second unique pathogen antigen and the first unique pathogenantigen are immunologically distinct but share conserved sites that arenot normally immunogenic for antibodies.

In another embodiment, the disclosure provides a use of a second uniquepathogen antigen for inducing a cross-protective antibody response in asubject that has been previously subjected to a first unique pathogenantigen 3-52 weeks prior, wherein the second unique pathogen antigen andthe first unique pathogen antigen are immunologically distinct but shareconserved sites that are not normally immunogenic for antibodies.

Also provided herein is a second unique pathogen antigen for use ininducing a cross-protective antibody response in a subject that has beenpreviously subjected to a first unique pathogen antigen 3-52 weeksprior, wherein the second unique pathogen antigen and the first uniquepathogen antigen are immunologically distinct but share conserved sitesthat are not normally immunogenic for antibodies.

Further provided herein is a use of a second unique pathogen antigen forpreparing a boost vaccine for vaccinating a subject that has beenvaccinated with a priming vaccine comprising a first unique pathogenantigen 3-52 weeks prior, wherein the second unique pathogen antigen andthe first unique pathogen antigen are immunologically distinct but shareconserved sites that are not normally immunogenic for antibodies.

In one embodiment, the pathogen antigen is a hemagglutinin (HA) proteinof influenza, a gB protein of human cytomegalovirus (HCMV), or a gp140or gp160 protein of human immunodeficiency virus (HIV) or a fragmentthereof.

In another embodiment, the method further comprises c) administering athird unique pathogen antigen 3-52 weeks after b), wherein the thirdunique pathogen antigen and the first and second unique pathogenantigens are immunologically distinct but share conserved sites that arenot normally immunogenic for antibodies.

In one embodiment, one or more additional unique pathogen antigens areused as a mixture or concurrently with the first and/or second uniquepathogen antigen.

Also provided herein is a method of inducing a cross-protective antibodyresponse in a subject against influenza comprising:

(a) administering a first unique hemagglutinin (HA) protein to thesubject; and

(b) administering a second unique HA protein 3-52 weeks after a),wherein the second unique HA protein and the first unique HA protein areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies.

In another embodiment, the disclosure provides a use of a second uniqueHA protein for inducing a cross-protective antibody response in asubject that has been previously subjected to a first unique HA protein3-52 weeks prior, wherein the second unique HA protein and the firstunique HA protein are immunologically distinct but share conserved sitesthat are not normally immunogenic for antibodies. The disclosure alsoprovides a second unique HA protein for use in inducing across-protective antibody response in a subject that has been previouslysubjected to a first unique HA protein 3-52 weeks prior, wherein thesecond unique HA protein and the first unique HA protein areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies. In yet another embodiment, the disclosureprovides a use of a second unique HA protein for preparing a boostvaccine for vaccinating a subject that has been vaccinated with apriming vaccine comprising a first unique HA protein 3-52 weeks prior,wherein the second unique HA protein and the first unique HA protein areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies.

In one embodiment, the second unique HA protein comprises a head that issubstantially antigenically unrelated to the first unique HA protein butcomprises conserved antigenic sites in the stem or on the head that arenormally not immunogenic for antibodies.

In an embodiment, the first and/or second unique HA protein is part ofan attenuated virus or inactivated virus. In another embodiment, thefirst or second unique HA protein is from a pandemic virus or a virusthat normally infects a different species of host. In one embodiment,the virus that infects a different host is a virus that infects an avianspecies or a virus that infects swine. In yet another embodiment, thefirst and/or second unique HA protein is an artificial HA protein havingmutations in the head that make it antigenically unrelated to HA thatthe population to be vaccinated has been exposed to or a chimeric HAprotein comprising a conserved stem coupled to a unique head.

In one embodiment, the first unique HA antigen is derived from anisolate of an H5N1 influenza A virus and the second unique HA antigen isderived from another avian influenza virus with a hemagglutinin of Group1, such as H1, H2, H5 (with the distinction that it should be an H5 of asubstantially different antigenicity) H6, H8, H9, H11, H12, H13, or H16optionally from a subtype of H2N9, H2N2, H6N1, H6N2, H8N4, and H9N2influenza A viruses.

Also provided herein is a method of inducing a cross-protective antibodyresponse in a subject against influenza comprising:

a) administering a first unique hemagglutinin (HA) protein that isderived from an isolate of an H5N1 influenza A virus; and

b) administering a second unique HA protein to the subject 3-52 weeksafter a), wherein the second unique HA protein derives from anotheravian influenza virus with a hemagglutinin of Group 1, such as H1, H2,H5, H6, H8, H9, H11, H12, H13, or H16 optionally from a subtype of H2N9,H2N2, H6N1, H6N2, H8N4, and H9N2 influenza A viruses.

In yet another embodiment, the first unique HA protein is derived from astrain or an isolate of H5N1 influenza A virus and the second unique HAprotein is derived from another mammalian influenza virus with ahemagglutinin of Group 1.

Also provided herein, is a method of inducing a cross-protectiveantibody response in a subject against influenza comprising:

(a) administering a first unique hemagglutinin (HA) protein to thesubject, wherein the first unique HA protein is from a strain or anisolate of H5N1 influenza A virus; and

(b) administering a second unique HA protein to the subject 3-52 weeksafter a), wherein the second unique HA protein is from another mammalianinfluenza virus with a hemagglutinin of Group 1. In one embodiment, theinfluenza virus with a hemagglutinin of Group 1 comprises a subtype ofinfluenza A virus circulating in pigs or horses.

In a further embodiment, the first unique HA protein is derived from astrain or an isolate of the 2009 H1N1 influenza A virus and the secondunique HA protein is derived from an isolate of an H5N1 influenza Avirus. Further provided herein is a method of inducing across-protective antibody response in a subject against influenzacomprising:

(a) administering a first unique hemagglutinin (HA) protein to thesubject, wherein the first unique HA protein is from a strain or anisolate of the 2009 H1N1 influenza A virus; and

(b) administering a second unique HA protein to the subject 3-52 weeksafter a), wherein the second unique HA protein is from an isolate of anH5N1 influenza A virus.

Also provided herein is a method of inducing a cross-protective antibodyresponse against influenza in a subject that has been vaccinated orinfected with an influenza virus that contains a first uniquehemagglutinin (HA) comprising administering to the subject a secondunique HA protein 3-52 weeks after the vaccination or infection, whereinthe second unique HA comprises a head that is substantiallyantigenically unrelated to the first unique HA, but exhibits conservedantigenic sites on the stem or conserved sites on the head that are notnormally immunogenic. Further provided is use of a second unique HAprotein for inducing a cross-protective antibody response againstinfluenza in a subject that has been vaccinated or infected with aninfluenza virus that contains a first unique hemagglutinin 3-52 weeksprior, wherein the second unique HA comprises a head that issubstantially antigenically unrelated to the first unique HA, butexhibits conserved antigenic sites on the stem or conserved sites on thehead that are not normally immunogenic. Even further provided is asecond unique HA protein for use in inducing a cross-protective antibodyresponse against influenza in a subject that has been vaccinated orinfected with an influenza virus that contains a first uniquehemagglutinin 3-52 weeks prior, wherein the second unique HA comprises ahead that is substantially antigenically unrelated to the first uniqueHA, but exhibits conserved antigenic sites on the stem or conservedsites on the head that are not normally immunogenic. Also provided isuse of a second unique HA protein in the manufacture of a medicament forinducing a cross-protective antibody response against influenza in asubject that has been vaccinated or infected with an influenza virusthat contains a first unique hemagglutinin 3-52 weeks prior, wherein thesecond unique HA comprises a head that is substantially antigenicallyunrelated to the first unique HA, but exhibits conserved antigenic siteson the stem or conserved sites on the head that are not normallyimmunogenic.

In one embodiment, the first unique HA protein is derived from the 2009H1N1 influenza A virus and the second unique HA protein is derived froman isolate of an H5N1 influenza A virus. Also provided herein is amethod of inducing a cross-protective immune response in a subjectagainst influenza that has been vaccinated or infected with the 2009H1N1 influenza A virus by administering to the subject, 3-52 weeks afterthe vaccination or infection a hemagglutinin (HA) that derives from anisolate of an H5N1 influenza A virus.

In another embodiment, the first unique hemagglutinin protein is a Group1 HA protein and the second unique hemagglutinin is a Group 2 HA proteinor the first unique hemagglutinin protein is a Group 2 HA protein andthe second unique hemagglutinin is a Group 1 HA protein.

In one embodiment, one or more additional unique HA proteins are used asa mixture or concurrently with the first and/or second unique HAprotein.

In yet a further embodiment, the unique first and/or secondhemagglutinin is coupled, such as by fusion, chemically or physically,to a T-cell peptide epitope that the subject has been previouslyimmunized against. Such T-cell peptide epitopes are well-known to thoseskilled in the art, for example, the epitope from tetanus toxoid.

Also provided herein is a kit comprising a first unique hemagglutinin(HA) protein as disclosed herein and a second unique hemagglutinin (HA)protein as disclosed herein, wherein the first unique HA protein and thesecond unique HA protein are immunologically distinct but shareconserved sites that are not normally immunogenic for antibodies.

In another embodiment, the present disclosure provides a method ofinducing a cross-protective antibody response in a subject against ahuman cytomegalovirus (HCMV) comprising:

(a) administering a gB glycoprotein or a fragment thereof comprising thegB ectodomain to the subject; and

(b) administering a modified gB glycoprotein 3-52 weeks after a);

wherein the modified gB glycoprotein lacks the AD-1 epitope.

In another embodiment, the disclosure provides a use of a modified gBglycoprotein for inducing a cross-protective antibody response in asubject that has been previously subjected to immunization with a gBglycoprotein or its ectodomain 3-52 weeks prior, wherein the modified gBglycoprotein lacks the AD-1 epitope.

The disclosure also provides a modified gB glycoprotein for use ininducing a cross-protective antibody response in a subject that has beenpreviously subjected to a first immunization with gB glycoprotein or itsectodomain 3-52 weeks prior, wherein the modified gB glycoprotein lacksthe AD-1 epitope.

In yet another embodiment, the disclosure provides a use of a modifiedgB glycoprotein for preparing a boost vaccine for vaccinating a subjectthat has been vaccinated with a priming vaccine comprising a gBglycoprotein or its ectodomain 3-52 weeks prior, wherein the modified gBglycoprotein lacks the AD-1 epitope.

In yet another embodiment the present disclosure provides a method ofinducing a cross-protective neutralizing antibody response in a subjectagainst HIV-1 comprising:

(a) administering a first unique gp140 or gp160 glycoprotein to thesubject; and

(b) administering a second unique gp140 or gp160 protein, 3-52 weeksafter a);

wherein the second unique gp140 or gp160 glycoprotein and the firstunique gp140 or gp160 glycoprotein are immunologically distinct butshare conserved sites that are not normally immunogenic for antibodies.

In another embodiment, the disclosure provides a use of a second uniquegp140 or gp160 glycoprotein for inducing a cross-protective antibodyresponse in a subject that has been previously subjected to immunizationwith a first unique gp140 or gp160 glycoprotein 3-52 weeks prior;wherein the second unique gp140 or gp160 glycoprotein and the firstunique gp140 or gp160 glycoprotein are immunologically distinct butshare conserved sites that are not normally immunogenic for antibodies.

The disclosure also provides second unique gp140 or gp160 glycoproteinfor use in inducing a cross-protective antibody response in a subjectthat has been previously subjected to immunization with a first uniquegp140 or gp160 glycoprotein 3-52 weeks prior; wherein the second uniquegp140 or gp160 glycoprotein and the first unique gp140 or gp160glycoprotein are immunologically distinct but share conserved sites thatare not normally immunogenic for antibodies.

In yet another embodiment, the disclosure provides a use of a secondunique gp140 or gp160 glycoprotein for preparing a boost vaccine forvaccinating a subject that has been vaccinated with a priming vaccinecomprising a first unique gp140 or gp160 glycoprotein 3-52 weeks prior,wherein the second unique gp140 or gp160 glycoprotein and the firstunique gp140 or gp160 glycoprotein are immunologically distinct butshare conserved sites that are not normally immunogenic for antibodies.

Further provided herein is an assay for detecting cross-protectiveantibodies in serum or plasma against an influenza virus comprising:

(a) incubating cells with the influenza virus and test serum sample for1-5 days; wherein the cells express a protease that cleaveshemagglutinin of the influenza; and

(b) detecting viral infectivity compared to a control without sample;

wherein a decrease in viral infectivity at 1-5 days indicates thepresence of cross-protective antibodies.

In one embodiment, the cells are from a mammalian cell line derived fromthe lung, airways or epithelial cells from the intestine.

Also provided herein are novel isolated complementarity determiningregions, light chain variable regions, heavy chain variable regions andantibodies and fragments thereof and methods and uses thereof forcross-protection against influenza infection or for treating infectionwith influenza.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will now be described in relation to the drawings inwhich:

FIG. 1 shows Biacore analysis of a human monoclonal antibody (mAb)binding to the recombinant ectodomain of the HA of the 2009 pandemicH1N1 influenza virus (snH1 HA). Anti-human IgG FC was covalently coupledto a Biacore Sensor Chip CM5 via primary amine groups. Purified mAbsI4-128 (A) and I4-1G8 (B) were diluted in HBS-P (10 mM HEPES, pH 7.4,150 mM NaCl, 0.01% Surfactant P-20) to 5 μg/ml and 750-800 RU of eachcaptured. Purified, recombinant ectodomain of the HA of nH1N1 (snH1 HA)was diluted in HBSP to 10 μg/ml and subsequently injected. Thesensorgram response (RU) shows the interaction of recombinant nH1 HAwith the captured antibodies. The snH1 HA was simultaneously injectedover a reference surface containing anti-human IgG FC and baselinesubtracted to give the resultant sensorgram.

FIG. 2 shows diverse cross-reactive/heterosubtypic anti-HA antibodiesare induced by infection or vaccination with nH1N1 and react withconventional vaccines at the HA head or stem and with HA from highlypathogenic avian H5N1 influenza A virus. A, Reactivity with nH1N1 orseasonal influenza vaccine and with purified recombinant ectodomain ofH5 HA (A/Vietnam/1203/2004, Clade1). ELISA titrations of selected mAbson plates coated with the nH1N1 vaccine or the 2009/2010 seasonalinfluenza vaccine or with purified recombinant H5 HA(A/Vietnam/1203/2004, Clade1). B, IGHV-gene usage in the 48 anti-snH1 HAmAbs. The 95% confidence intervals of IGHV1-69 usage were 38-66%. *8 of9 mAbs using IGHV4-39 were from a single subject, V2. C, Somecross-reactive/heterosubtypic antibodies bind to the HA stem others tothe HA head. Above panel:—competition ELISA of selected mAbs on nH1N1vaccine showing inhibition of binding of C179, which recognizes anepitope on the HA stem. Circles: I5-24 (open); I4-128 (filled).Triangles: I8-1B6 (upright, open), I5-52 (inverted, filled). Squares:V4-17 (filled); Control mAb, KE5 against HCMV (open). Belowpanel:—treatment of nH1N1-coated ELISA plates at pH 5 for 1 h prior tomAb incubation causes inhibition of binding by IGHV1-69-encoded mAbs. D,IGHV1-69-using mAbs V3-2G6, I5-24, V3-1G10 and I8-1B6 fail to inhibithemagglutination by nH1N1 but the IGHV4-39-using mAb V2-36 does andV4-12 does too. The indicated mAbs (40 μg/mL) were titrated and a valueof zero means that hemagglutination was not inhibited at a titre of <2or 20 μg/mL. E,F Cross-reactivity of selected mAbs with the HA ofinfluenza A/Hong Kong/156/197 (H5N1). Indirect immunofluorescence wasused to assess the binding of mAbs to H5 HA expressed by adenovirusinfection in the A549 human epithelial cell-line using a Cellomicsinstrument. E, Shows instrument-image of cells stained with eithersecondary antibodies alone or 5 μg/mL of mAb I4-128. F, Shows thepercentage of cells stained with varying concentrations of the indicatedmAbs or titres of plasma from an nH1N1-vaccinated subject (V2) or annH1N1-infected subject (I14). Shown is the average of duplicatemeasurements from a representative experiment of three.

FIG. 3 shows neutralization of multiple subtypes of influenza A virusesby mAbs. A, Neutralization of nH1N1 by the indicated purified mAbs thatwere present for the entire assay. Titrations commenced at aconcentration of 5 μg/mL of each mAb. * indicates titre >256. B,Neutralization of infectivity of highly pathogenic InfluenzaA/Goose/Ger/R1400/07 (H5N1) avian virus by indicated mAbs and bytitrations of plasma from subjects infected (I14) or vaccinated (V2)with nH1N1. Also shown below is a graph of the neutralization oftitrations of selected mAbs against highly pathogenic InfluenzaA/Goose/Ger/R1400/07 (H5N1) avian virus and with below a photograph ofthe plaques with and without an anti-HA stem mAb I8-1B6 (1 μg/ml). C,Anti-stem mAbs encoded by IGHV1-69 inhibit H5 HA-mediated fusion. Notethe decreased syncytia formation in wells treated by the anti-stem mAb,C179, and the IGHV1-69-using mAbs, in comparison to the control with noantibody added (no Ab) and a mAb that binds to the HA head, V2-36, wherethere are many syncytia.

FIG. 4 shows IGHV1-69 encoded monoclonal antibodies do not neutralizeplaque formation by A/Ck/Ger/R28/03 (H7N7) influenza A viruses at 4μg/mL.

FIG. 5 shows evidence that some of the cross-reactive/heterosubtypicmAbs originated from memory B cells that were activated by nH1N1. A,Numbers of somatic mutations in IGHV-genes of mAbs generated fromplasmablasts and B memory cells. Mann-Whitney Rank Sum Test of thenumber of somatic mutations in IGHV genes of mAbs generated from memoryB cells (N=22) versus plasmablasts (N=22) demonstrates a significantdifference (P=0.001). Whiskers show the 5/95th percentile. B,Pre-existing titres of antibodies binding to cells expressing the HA ofinfluenza A/Hong Kong/156/197 (H5N1), quantified as in FIGS. 1D and 1E,increase in the plasma of a subject 7 days after vaccination with nH1N1.

FIG. 6 shows results of treatment of CD-1 mice infected intranasallywith a lethal dose (2×10⁵ plaque-forming units) of the 2009 novel H1N1influenza A virus (nH1N1) with the indicated doses of the humanmonoclonal antibody V2-36 expressed as IgG1. The mice were injected onceintraperitoneally with the mAb 24 hours after infection intranasallywith the pandemic nH1N1 virus. Control mice were injected with diluentalone (phosphate-buffered saline, PBS). The mice were weighed daily.

FIG. 7 shows results of treatment of groups of 5 CD-1 mice infectedintranasally with a lethal dose (2×10⁵ plaque-forming units) of the 2009novel H1N1 influenza A virus with the indicated human monoclonalantibodies expressed as IgG1 molecules. With the exception of V2-36(labeled in the FIG. 7 as BC002-36, later named V2-36 as it wasgenerated from a subject vaccinated with the pandemic H1N1 vaccine)which was given as a dose of 200 μg intraperitoneally 48 hours afterinfection, all mAbs were given at a dose of 300 μg intraperitoneally 24hours after infection. The mAbs labelled in the FIG. 7 as BC003-2G6 andBC003-1G10, were later named V3-2G6 and V3-1G10 as they were generatedfrom a subject vaccinated with the pandemic H1N1 vaccine and 8-1B6,4-128 and 5-24 were later named I8-1B6, I4-128 and I5-24, as they wereall generated from subjects infected with the pandemic H1N1 influenzavirus. The mixture of 100 μg of V2-36 and 150 μg of I5-24 was given 24hours after infection. Controls were injected with vehicle alone (PBS).Results are shown per treated group of 5 as survival rate or in terms ofaverage weight. Shown is the result of one experiment, for clarity splitinto two panels.

FIG. 8 shows therapeutic efficacy of a human monoclonal heterosubtypicantibody generated from a human vaccinated with the pandemic H1N1vaccine in a lethal infection of BALB/c mice with heterologous H5N1influenza/A virus. A. Groups of 5 mice were infected intranasally with2×10⁵ PFU of A/Hong Kong/213/2003 (H5N1) on the PR8/34 virus backbone,and treated after 24 hrs with 150 or 300 μg of V3-2G6 or after 48 hrswith 300 μg or 600 μg of V3-2G6. Controls were treated withPhosphate-buffered saline (PBS). Survival and weight-loss was monitored.B. Further reduction of the therapeutic dose of V3-2G6 to enable mice tosurvive from a lethal infection with H5N1. Groups of 5 mice wereinfected intranasally with 3×10⁵ PFU of A/Hong Kong/213/2003 (H5N1) onthe PR8/34 virus backbone, and treated after 24 hrs with 150 μg, 75 μgor 37.5 μg of V3-2G6. Note that all treated mice survived but there wasa prolonged weight loss in the group treated with the least dose.

FIG. 9 shows cross-protection against heterologous lethal infection inmice by H5N1 influenza virus conferred by a heterosubtypic monoclonalantibody generated from a human vaccinated with the pandemic nH1N1vaccine. Twenty-four hours before intranasal infection with 2×10⁵ PFU ofA/Hong Kong/213/2003 (H5N1) virus, 3 groups of 5 BALB/c mice wereinjected intraperitoneally with PBS as a control, or 15 μg or 5 μg ofV3-2G6 generated from a subject vaccinated with the pdmH1N1 vaccine.Data shows survival rates and average weight at over 14 days.

FIG. 10 shows cross-protection against heterologous lethal infection inmice by H5N1 influenza virus conferred by plasma from a human collectedat two weeks and a year after vaccination with the pandemic H1N1vaccine. Twenty-four hours before intranasal infection with 2×10⁵ PFU ofA/Hong Kong/213/2003 (H5N1) virus, groups of BALB/c mice were treatedwith a 400 μl intraperitoneal injection of PBS as a control or plasmacollected from subject V3 either 14 days post pdmH1N1 vaccination or 1year post pdmH1N1 vaccination and as a control, plasma from a youngindividual collected before 2009 and thus unvaccinated and uninfectedwith the pandemic H1N1 influenza virus. Another group of mice werepre-treated 72, 48 and 24 hrs prior to infection with 400 μl of plasmafrom V3 collected 1 year after vaccination with the pandemic H1N1vaccine.

DETAILED DESCRIPTION OF THE DISCLOSURE

Methods and Uses for Protecting Against Pathogen Infection

The present inventor has demonstrated that a protective antibodyresponse can be induced against a desired, conserved, functional site ona pathogen or pathogen antigen, where that site is not normallyimmunogenic or is minimally immunogenic because of competition in theimmune response for T cell help from B cells making antibodies againstother sites on the pathogen antigen. For example, the protective humanantibody response to influenza is normally very isolate/strain-specificbecause the human immune response does not normally make a vigorousprotective antibody response against sites on the virus that areconserved between strains. The present inventor has shown that this isbecause, in the antibody response, many memory B cells specific forother sites on the antigen, compete for T-cell help. The methodsdisclosed herein avoid re-stimulating the memory B cells makingantibodies to readily immunogenic parts of the immunogen and thus avoidthe competition between B cells that normally impedes the development ofan antibody response to the conserved site, providing cross-protectionagainst different strains and subtypes of influenza viruses.

In contrast to immune focusing by practising on naïve animals withsequential immunizations with four hemagglutinins of the same subtypeH3, where a small percentage of 120 monoclonal antibodies were shown toprotect against multiple H3N2 viruses circulating in humans (Wang et al2010), the present disclosure provides methods and uses designed toelicit protective levels of cross-protective heterosubtypic antibodiesin individuals who already have immunity to an influenza. The presentmethods/uses of sequences of unique hemagglutinins avoids stimulatingmemory B-cells against sites that the virus can readily vary and enablesheterosubtypic antibodies that reach protective levels.

For the purpose of vaccinating a naïve human or animal to a variablepathogen to induce broadly protective antibodies, after the firstimmunization (which will be by definition with a unique antigen sincethe animal is naïve), the second and subsequent immunizations should beall with further distinct unique antigens, such that the subject to bevaccinated had undetectable or very few memory B cell making antibodiesthat bind tightly to these further unique antigens.

In particular, the present disclosure is based on the presentdemonstration that antibodies that bind to a particular site on anantigen, for example an epitope on a protein, can be difficult to raisebecause of competition by the relatively greater number of memory Blymphocytes that make antibodies specific for other sites on theantigen. This arises because the B lymphocytes that make the antibodiesagainst the desired site on the antigen are less numerous than the totalnumber of B lymphocytes that make antibodies against other sites on theantigen. These more numerous B lymphocytes specific for other epitopeson the antigen will out-compete the B lymphocytes specific for thedesired epitope for resources in the lymph node and germinal centre, inparticular the helper T lymphocytes specific for epitopes carried by theantigen or proteins associated with it (Allen et al, 2007, Victora, etal 2010, Schwickert et al 2011). For example, in humans, due to repeatedencounters with seasonal influenza, there is a great number andfrequency of B lymphocytes that make antibodies against the head of theseasonal influenza hemagglutinin molecule (Corti et al, 2010 andWrammert et al, 2008, 2011) out-compete the less numerous B lymphocytesthat make antibodies against the stem of the hemagglutinin that, unlikemost of those against the head, cross-react with and neutralize manystrains of influenza. Extremely low affinities are sufficient toactivate memory B cells and prepare them to enter the germinal centre(Schwickert et al, 2011). In an infection or vaccination with a seasonalinfluenza virus that has undergone “antigenic drift”, there will be manymemory B cells induced by the HA head of the original virus that wouldstill have low affinity for and be activated by the HA of the “drifted”virus. This means that the memory B cells against the original HA headwould enter the germinal centres and undergo another round of affinitymaturation and acquire somatic mutations that will increase the affinityfor the stimulating “drifted” HA. Those memory B cells activated by lowaffinity interactions with the drifted HA would outcompete for T cellhelp those rare memory B cells against the HA stem. In contrast with thelow affinity required for B cells to acquire antigen and present antigento T cells, the affinity/avidity of antibodies against the HA headneeded to block viral attachment to the host cell and thus neutralizeinfectivity is relatively high (Knossow et al, 2002). This means thatalthough viruses or vaccines with drifted HA can still activate memory Bcells and enter germinal centres (Wrammert et al 2008), they are notneutralized by the antibodies induced by the original virus.

In addition, memory B cells have advantages over naïve B cells incompeting for T cell help. Thus memory B cells are more easily activatedby antigen because they signal more efficiently than naïve B cells. Theyalso express more proteins that co-stimulate T cells than naïve B cellsand also have different migration patterns. For these reasons memory Bcells, even those that cross-react weakly with an antigen on a pathogenthat has been mutated to avoid neutralization by existing antibodies,can still have a competitive advantage over naïve B cells that make anantibody specific for the mutation.

Accordingly, the present disclosure provides a method of inducing across-protective antibody response in a subject against a pathogencomprising:

(a) administering a first unique pathogen antigen to the subject; and

(b) administering a second unique pathogen antigen 3-52 weeks,optionally 4-16 weeks, after a), wherein the second unique pathogenantigen and the first unique pathogen antigen are immunologicallydistinct but share conserved sites that are not normally immunogenic forantibodies.

In another embodiment, the disclosure provides a use of a second uniquepathogen antigen for inducing a cross-protective antibody response in asubject that has been previously subjected to a first unique pathogenantigen 3-52 weeks, optionally 4-16 weeks, prior, wherein the secondunique pathogen antigen and the first unique pathogen antigen areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies.

Also provided herein is a second unique pathogen antigen for use ininducing a cross-protective antibody response in a subject that has beenpreviously subjected to a first unique pathogen antigen 3-52 weeks,optionally 4-16 weeks, prior, wherein the second unique pathogen antigenand the first unique pathogen antigen are immunologically distinct butshare conserved sites that are not normally immunogenic for antibodies.

Further provided herein is a use of a second unique pathogen antigen forpreparing a boost vaccine for vaccinating a subject that has beenvaccinated with a priming vaccine comprising a first unique pathogenantigen 3-52 weeks, optionally 4-16 weeks, prior, wherein the secondunique pathogen antigen and the first unique pathogen antigen areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies.

The term “pathogen” as used herein refers to a microorganism that causesa disease, including without limitation, a virus, bacterium, fungus orparasite.

In one embodiment, the pathogen antigen is a hemagglutinin (HA) proteinof the influenza virus or a fragment thereof. In another embodiment, thepathogen antigen is a HCMV protein, for example, the gB glycoprotein, ora fragment thereof. In yet another embodiment, the pathogen antigen isan HIV protein, for example, the gp140 ectodomain of the gp160glycoprotein. Methods and uses related to these pathogen antigens arefurther described herein.

In another embodiment, where there is existing immunity against theconserved sites on the pathogen, the administration of (a) the firstunique antigen is unnecessary and only a single administration, as in(b), is used. In one embodiment, there is provided a use of a uniquepathogen antigen for inducing a cross-protective antibody response in asubject that has existing immunity against the conserved sites on thepathogen. Also provided is a unique pathogen antigen for use in inducinga cross-protective antibody response in a subject that has existingimmunity against the conserved sites on the pathogen. Further providedis a use of a unique pathogen antigen in the manufacture of a medicamentfor inducing a cross-protective antibody response in a subject that hasexisting immunity against the conserved sites on the pathogen.

In yet another embodiment, the method further comprises: (c)administering a third unique pathogen antigen 3-52 weeks, optionally4-16 weeks, after b), wherein the third unique pathogen antigen and thefirst and second unique pathogen antigens are immunologically distinctbut share conserved sites that are not normally immunogenic forantibodies. Also provided herein is a use of a third unique pathogenantigen for inducing a cross-protective antibody response in a subjectthat has been previously subjected to a second unique pathogen antigen3-52 weeks, optionally 4-16 weeks, prior and previously has beensubjected to a first unique pathogen 3-52 weeks, optionally 4-16 weeks,prior to the second unique pathogen antigen, wherein the third uniquepathogen antigen and the first and second unique pathogen antigens areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies. Further provided is a third unique pathogenantigen for use in inducing a cross-protective antibody response in asubject that has been previously subjected to a second unique pathogenantigen 3-52 weeks, optionally 4-16 weeks, prior, and previously hasbeen subjected to a first unique pathogen 3-52 weeks, optionally 4-16weeks, prior to the second unique pathogen antigen, wherein the thirdunique pathogen antigen and the first and second unique pathogenantigens are immunologically distinct but share conserved sites that arenot normally immunogenic for antibodies. Further provided herein is ause of a third unique pathogen antigen for preparing a boost vaccine forvaccinating a subject that has been vaccinated with a priming vaccinecomprising a second unique pathogen antigen 3-52 weeks, optionally 4-16week, and previously has been subjected to a first unique pathogen 3-52weeks, optionally 4-16 weeks, prior to the second unique pathogenantigen, wherein the third unique pathogen antigen and the first andsecond unique pathogen antigens are immunologically distinct but shareconserved sites that are not normally immunogenic for antibodies.

The phrase “wherein the first and second unique pathogen antigens areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies” as used herein means that the first uniquepathogen antigen elicits antibodies that do not generally bind stronglyto the second unique pathogen antigen except for conserved sites thatare shared between the antigens that are not normally immunogenic or areminimally immunogenic due to competition for T cell help by the morenumerous B cells stimulated by the sites that are normally immunogenic.Similarly if a third unique pathogen antigen is used, it isimmunologically distinct from the first and second unique pathogenantigen but shares conserved sites that are not normally immunogenic forantibodies.

The term “unique pathogen antigen” or “unique protein” as used hereinrefers to a pathogen antigen or fragment thereof that is unique orunfamiliar to the subject it is desired to vaccinate, so that there areno or few memory B cells activated by contact with the “unique pathogenantigen” and there are no or low levels of antibodies circulating in thesubject that bind to the unique pathogen antigen. In the “uniquepathogen antigen”, epitopes that typically elicit a strong antibodyresponse in the subject are different, for example, due to mutation ordeletion of amino acids or different glycosylation, from epitopes onrelated pathogen antigens previously encountered by the subject, whetherby natural infection or vaccination. However, the “unique pathogenantigen” shares common B-cell epitopes that are the target of protectiveantibodies in the subject but that are normally not immunogenic becauseof competition for T cell help by the more numerous B cells stimulatedby immunogenic parts of the pathogen antigen. The low frequency ofmemory B cells in the population to be vaccinated induced by the uniquehemagglutinin that make antibodies that bind tightly to the putativeunique antigen can be readily ascertained using the methods of Wen et al1987 or Corti et al 2010. In the population to be vaccinated, thefrequency of memory B cells making antibodies reacting strongly to theunique antigen is undetectable or less than 1 per million, or at leastless than 0.01% of IgG-expressing memory B cells circulating in theblood, as ascertained using the methods of Wen et al 1987 or Corti et al2010.

The term “pathogen antigen” as used herein refers to a pathogen orcomponent thereof that elicits an immune response in the subject andincludes, without limitation, proteins or sugars or lipids or fragmentsthereof or combinations thereof, expressed by the pathogen.

The phrase “sites that are not normally immunogenic” as used hereinrefers to sites that do not elicit a strong antibody response due tocompetition for T cell help by the more numerous B cells that areactivated by other sites on the pathogen or pathogen antigen andincludes sites that are minimally immunogenic in subjects that have beenrepeatedly exposed to strains of the pathogen and account for less than5% of the antibody response against that pathogen antigen.

The term “induces a cross-protective antibody response” as used hereinmeans that administration of an effective dose of the first uniquepathogen antigen, followed by the second unique pathogen antigen asdescribed herein, results in the production of antibodies that inhibitor reduce the severity of infection by multiple different pathogenstrains or subtypes, for example, by avoiding the activation of animmunodominant population of memory B cells that make antibodies againstanother site on the pathogen that do not cross-protect.

To minimize competition by memory B cells activated by immunogenic sitesin the first pathogen antigen that still persist in the lymph-nodes fromthe first administration at the time of the second administration, thesecond pathogen antigen optionally is administered or used at adifferent site.

Administration at a different site is also useful to stimulatecross-reactive, cross-protective antibody responses to various strainsof a pathogen and, at the same time, to increase the levels ofstrain-specific antibodies to a particular strain of a pathogen. Forexample in the aged, the response to seasonal influenza vaccine isdiminished. In this case, the present methods are useful for increasingthe levels of cross-reactive, heterosubtypic cross-protective antibodylevels to influenza by exploiting the cross-reactive heterosubtypicmemory B cells built up by years of contact with influenza. However itmay also be advantageous to also stimulate at the same time, thestrain-specific antibody response to a particular strain or subtype ofan influenza virus, for example the currently circulating H3N2 influenzavirus. In this case the particular pathogen antigen that will inducestrain-specific antibodies should be administered in a different sitefrom the specified, unfamiliar or unique antigen designed to inducecross-protective antibodies—for example the first administration in oneshoulder and the second administration in the opposite shoulder.

Accordingly, in another embodiment, the administration in b) is at adifferent anatomical site from the administration in a).

Administration of a pathogen antigen includes, without limitation,administration of a fragment of the pathogen antigen, or administrationof the pathogen antigen as a component of a virus, optionallyinactivated by methods known to those skilled in the state of the artsuch as a “split” vaccine plus or minus a suitable adjuvant known tothose skilled in the state of the art, or a virus comprising the uniquepathogen antigen that has been attenuated by methods known to thoseskilled in the state of the art or a viral-like particle comprising theunique pathogen antigen generated by methods known to those skilled inthe state of the art or a DNA or RNA vector encoding the unique pathogenantigen using methods known to those skilled in the state of the art.

In yet another embodiment, one or more additional unique pathogenantigens are used as a mixture or concurrently with the first and/orsecond unique pathogen antigen. For example, the first unique pathogenantigen could be mixed with an additional unique pathogen antigen or thefirst unique pathogen antigen could be administered at one anatomicalsite and the additional unique pathogen antigen could be administered ata second anatomical site concurrently (i.e. at the same time or closetogether, e.g. within 24 hours). The second unique pathogen antigenwould then be administered 3-52 weeks after the first administration andcould also be mixed with one or more additional unique pathogen antigensor administered concurrently with one or more additional unique pathogenantigens.

Influenza Viruses

The present inventor has demonstrated that the immune response to the HAprotein in humans infected or vaccinated with nH1N1, unlike the responseto seasonal influenza, was dominated by antibodies that cross-reactedwith the HA of other strains or sub-types of influenza virus(“cross-reactive” or “heterosubtypic”), including highly pathogenic H5N1avian influenza, and could neutralize their infectivity. Moreover,antibodies that bound exclusively to the HA of nH1N1 were rare and onlyone out of 48 mAbs that bound the HA of nH1N1, failed to react withseasonal influenza vaccine (Table 1). About half (52%) of thecross-reactive or heterosubtypic antibodies used the IGHV1-69 gene (FIG.2), which encodes the major features of a binding site for a region onthe HA stem that is conserved in many subtypes of influenza virus(Ekiert et al. 2009; Sui et al. 2009). Moreover, there were 6% moreheterosubtypic antibodies against the HA stem that did not use IGHV1-69.The large numbers of somatic mutations in many of these antibodies(Table 1, FIG. 5) indicated that their origin was in memory B cells,presumably induced by seasonal influenza. While most of thecross-reactive or heterosubtypic antibodies generated against the uniqueHA of the nH1N1 virus bound to the stem of the HA, most antibodies fromone particular vaccinated individual (V2) were directed against the headof the HA as shown by the fact that they inhibited hemagglutination(e.g. V2-36, FIG. 2). The absence of memory B cells that could beactivated by the antigenically unique HA head of nH1N1 enabled therelatively rare memory B cells against conserved sites on the nH1N1 HAto effectively compete for T-cell help against naïve B cells and todominate the response. The demonstration that the nH1N1 influenzainduces the production of cross-protective antibodies, establishes thefeasibility of vaccination strategies for broad-spectrum protectionagainst influenza.

Accordingly, in an embodiment, the present disclosure provides a methodof inducing a cross-protective antibody response in a subject againstinfluenza comprising:

(a) administering a first unique hemagglutinin (HA) protein to thesubject; and

(b) administering a second unique HA protein 3-52 weeks, optionally 4-16weeks after a), wherein the first and second unique HA proteins areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies.

In another embodiment, the disclosure provides a use of a second uniqueHA protein for inducing a cross-protective antibody response in asubject that has been previously subjected to a first unique HA protein3-52 weeks, optionally 4-16 weeks prior, wherein the first and secondunique HA proteins are immunologically distinct but share conservedsites that are not normally immunogenic for antibodies. The disclosurealso provides a second unique HA protein for use in inducing across-protective antibody response in a subject that has been previouslysubjected to a first unique HA protein 3-52 weeks, optionally 4-16 weeksprior, wherein the first and second unique HA proteins areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies. In yet another embodiment, the disclosureprovides a use of a second unique HA protein for preparing a boostvaccine for vaccinating a subject that has been vaccinated with apriming vaccine comprising a first unique HA protein 3-52 weeks,optionally 4-16 weeks prior, wherein the first and second unique HAproteins are immunologically distinct but share conserved sites that arenot normally immunogenic for antibodies.

The term “hemagglutinin protein” or “HA protein” as used herein refersto a protein found on the surface of influenza viruses that helps theviruses attach to receptors. A hemagglutinin protein has both a headregion and a stem region. The term “hemagglutinin protein” also refersto fragments or components of the HA protein that are capable ofeliciting the desired antibody response, such as the ectodomain of HA,and includes, without limitation, both protein and sugar components thatare able to elicit or modify an antibody response.

The term “unique HA protein” as used herein refers to an HA protein thatis unfamiliar or unique to the subject it is desired to vaccinate orinduce a cross-protective antibody response in, so that there are no orfew memory B cells activated by contact with the “unique HA protein” andthere are no or low levels of antibodies circulating in the subject thatbind to the unique HA protein. In the “unique HA protein” epitopes thattypically elicit a strong antibody response in the subject aredifferent, for example, due to mutation or deletion of amino acids ordifferent glycosylation, from epitopes on related HA proteins previouslyencountered by the subject, whether by natural infection or vaccination.However, the “unique HA protein” shares common B-cell epitopes that arethe target of protective antibodies but that are normally notimmunogenic because of competition for T cell help by the more numerousB cells stimulated by immunogenic parts of the HA protein.

Unique hemagglutinins thus include, without limitation, thehemagglutinins of influenza virus strains that do not normally infectthe species of the subject, for example like that of the H5N1 avianinfluenza virus for human subjects, or for example, in humans,hemagglutinins that are antigenically distinct and have littlereactivity with antibodies against the strains of H1N1 and H3N2 seasonalinfluenza viruses which have been circulating in humans. An example of aunique HA in most of the human population is the HA of the 2009 pandemicinfluenza H1N1 strain as many humans had no circulating antibodies thatcould bind to it and neutralize its infectivity (Itoh et al. 2009) andthus lacked the memory B cells that could be activated by thehemagglutinin head or made antibodies of low affinity with the head ofthe hemagglutinin of the nH1N1 such that they could not compete againstrare memory B cells making cross-protective antibodies against aconserved site on the hemagglutinin stem.

The published literature indicates that the cross-reactive antibodiesagainst the H5 HA stem region fail to cross-react with H3 or H7hemagglutinins (Sui et al. 2009). However there is evidence thatantibodies can be induced in mice, which cross-react with a variety ofH3 influenza viruses (Wang et al. 2010). Therefore the methods and usesdisclosed herein can be practiced to induce antibodies which cross-reactwith a variety of H3 influenza by ensuring that subjects areadministered an H3 HA that is novel to that population. Unique H3 HAinclude, without limitation, an H3 influenza virus that circulated manyyears ago and which sera from the population does not display reactivityto, an H3 influenza virus that was circulating in another speciesindependent of human contact, and a mutated or chimeric HA thatexhibited the stem of the H3 HA but that lacked epitopes on the headthat the sera from the population to be vaccinated reacted with.

Also the literature indicates that there are conserved epitopes on thehemagglutinin head that can cross neutralize influenza A viruses fromdifferent subtypes and across group 1 and Group 2 subtypes ofhemagglutinin (Yoshida et al, 2009). Therefore by sequentiallyimmunizing with unique hemagglutinins from Group 1 and/or Group 2 ofhemagglutinins, broadly cross-protective antibodies will be induced.

In all cases, these hemagglutinins are “unique” in the sense that theylack B cell epitopes that are strongly immunogenic in the subject to bevaccinated because they cannot activate many memory B cells in thesubjects to be vaccinated and do not bind, or bind to only low levelswith low affinity to antibodies circulating in the subjects to bevaccinated. Nevertheless they exhibit conserved epitopes that arecritical for function, for example on the HA stem that support fusion.Optionally these unique hemagglutinins preserve the trimeric structure.

The term “cross-protective antibody response” as used herein refers toeliciting an antibody response to multiple strains of pathogens, such asinfluenza, strains and/or subtypes.

The term “antigenic sites” as used herein refers to sites on the HAprotein capable of eliciting an immune response. Thus, “conservedantigenic sites” refers to sites present in both the first and secondunique HA protein.

Administration of a hemagglutinin (HA) includes without limitation,administration of at least the ectodomain of the hemagglutinin, oradministration of the hemagglutinin as a component of a virus,optionally inactivated by methods known to those skilled in the state ofthe art such as a “split” vaccine plus or minus a suitable adjuvantknown to those skilled in the state of the art, or a virus comprisingthe unique HA that has been attenuated by methods known to those skilledin the state of the art or a viral-like particle comprising the uniqueHA generated by methods known to those skilled in the state of the artor a DNA or RNA vector encoding the unique HA using methods known tothose skilled in the state of the art.

Accordingly, in one embodiment, the first and/or second unique HAprotein is from a pandemic virus or a virus that normally infects adifferent host species. In one embodiment, the virus that infects adifferent host species is a virus that infects an avian species or avirus that infects swine.

In another embodiment, the first and/or second unique HA protein is partof an attenuated or inactivated influenza virus strain.

The term “inactivated influenza virus strain” as used herein refers toan influenza virus strain that is not infectious. For example, aninfluenza virus can be inactivated by dilute formaldehyde orbeta-propiolactone followed by a detergent treatment called splitting(Bardiya and Bae, 2005).

The term “attenuated influenza virus strain” as used herein refers to avirus that is live but has reduced virulence. Methods of rendering alive virus less virulent are known in the art and involve coldadaptation or other methods (Bardiya and Bae, 2005). An advantage oflive viruses is that they can be administered via nasal insufflationand/or at lower concentrations of virus rendering large-scaleinoculations less expensive. Live virus for example elicits diverseand/or heightened immune responses in the recipient of the HA protein,including for example systemic, local, humoral and cell-mediated immuneresponses.

Alternatively, the first and/or second unique HA protein is anartificial HA protein, optionally in a form that preserves the trimericectodomain of HA. Artificial HA proteins are designed to includemutations in the surface of the HA head that make it antigenicallyunrelated to HA that the population to be vaccinated has been exposedto. Such proteins may be expressed by a vector comprising DNA sequencesthat encode the HA protein.

One method of artificially generating a unique hemagglutinin is tomutate surface exposed residues on the head of the hemagglutinin of aseasonal influenza virus strain to disrupt the existing B cell epitopes,while maintaining the antigenic sites on the stem.

In yet another embodiment, the first and/or second unique HA protein isa chimeric protein, which comprises a conserved stem coupled to a headof a unique HA protein.

In one embodiment, artificial or chimeric HA proteins comprise T-cellepitopes, optionally linked to the HA protein by fusion, chemically orphysically, that the subject has been immunized against, for example, apeptide from tetanus toxoid. In another embodiment, where the wholevirus, inactivated or attenuated, is administered, the T cell help isprovided by epitopes on other proteins in the virion.

In yet a further embodiment, the second unique HA protein comprises ahead that is substantially antigenically unrelated to the first uniqueHA protein but comprises conserved antigenic sites in the stem or headthat are normally not immunogenic for antibodies. The term“substantially antigenically unrelated” as used herein means that sitesor epitopes on the first and second unique HA protein that normallyelicit a strong antibody response in the subject are different forexample of a different HA subtype from the first unique antigen suchthat subjects to be vaccinated or subjects vaccinated with step a) havesera that do not neutralize in vitro the infectivity of an influenzavirus exhibiting the unique hemagglutinin by the WHO standard assay, orhave an hemagglutination inhibition assay titre against an influenzavirus exhibiting the unique hemagglutinin of less than 40. The antigensare designed so that the second unique antigen does not activate manymemory B cells against the head induced by the first unique antigen. Thelow frequency of memory B cells induced by the first the uniquehemagglutinin that make antibodies that bind tightly to the secondputative unique antigen can be readily ascertained using the methods ofWen et al 1987 or Corti et al 2010.

In another embodiment, the first unique hemagglutinin protein is amember of Group 1 subtypes of HA protein and the second uniquehemagglutinin is a member of a Group 2 subtypes of HA protein or thefirst unique hemagglutinin protein is a Group 2 HA protein and thesecond unique hemagglutinin is a Group 1 HA protein. The term “Group 1HA” as used herein refers to the hemagglutinins of the subtypes H1, H2,H5, H6, H8, H9, H11, H12, H13 and H16. The term “Group 2 HA” as usedherein refers to the hemagglutinins of the subtypes H3, H4, H7, H10, H14and H15.

In yet another embodiment, one or more additional unique HA proteins areused as a mixture or concurrently with the first and/or second unique HAprotein. For example, the first unique hemagglutinin protein could be aGroup 1 HA protein and could be mixed with a Group 2 HA protein or theGroup 1 HA protein could be administered at one anatomical site and theGroup 2 HA protein at a second anatomical site concurrently (i.e. at thesame time or close together, e.g. within 24 hours). The second uniquehemagglutinin protein would then be administered 3-52 weeks after thefirst administration and could also be mixed with one or more additionalunique HA proteins or administered concurrently with one or moreadditional unique HA proteins.

Also provided herein is a kit comprising a first unique hemagglutinin(HA) protein as disclosed herein and second hemagglutinin (HA) proteinas disclosed herein, wherein the first HA protein and the second HAprotein are immunologically distinct but share conserved sites that arenot normally immunogenic for antibodies. In one embodiment, the first HAprotein comprises a head that is different from the second HA proteinand the first HA protein and the second HA protein comprise a stemhaving conserved antigenic sites that are not normally immunogenic.

In one embodiment the kit is used for inducing a cross-protectiveantibody response in a subject.

In an embodiment, the kit further comprises an instrument foradministering the HA proteins and/or instructions for use and/or acontainer.

Human Cytomegaloviruses (HCMV)

There is also a great need for a vaccine that will protect against HCMV.Antibodies against a linear epitope on the gB glycoprotein termed theAD-2 epitope can neutralize infectivity of multiple strains of HCMV.However, antibodies against AD-2 are generated in only a minority ofhumans vaccinated with previous vaccines and are not generated even inmany infected humans. The present inventor has shown that humanantibodies against AD-2 have to have a very specific structure and areusually derived from a single pair of immunoglobulin V-genes (McLean etal. 2005; McLean et al. 2006; Thomson et al. 2008); these structuralconstraints mean that B lymphocytes specific for AD-2 are relativelyinfrequent as compared with B lymphocytes that are specific for anotherepitope on the ectodomain of the gB protein, AD-1. The methods describedherein allow for decreased competition from AD-1 specific B lymphocytes.

Accordingly, in another embodiment, the present disclosure provides amethod of inducing a cross-protective antibody response in a subjectagainst human cytomegalovirus (HCMV) comprising:

(a) administering a gB glycoprotein or a fragment thereof comprising thegB ectodomain to the subject; and

(b) administering a modified gB glycoprotein 3-52 weeks, optionally 4-16weeks, after a), wherein the modified gB glycoprotein lacks the AD-1epitope.

In another embodiment, the disclosure provides a use of a modified gBglycoprotein for inducing a cross-protective antibody response in asubject that has been previously subjected to a gB glycoprotein or itsectodomain 3-52 weeks, optionally 4-16 weeks prior, wherein the modifiedgB glycoprotein lacks the AD-1 epitope. The disclosure also provides amodified gB glycoprotein for use in inducing a cross-protective antibodyresponse in a subject that has been previously subjected to a gBglycoprotein or its ectodomain 3-52 weeks, optionally 4-16 weeks prior,wherein the modified gB glycoprotein lacks the AD-1 epitope. In yetanother embodiment, the disclosure provides a use of a modified gBglycoprotein for preparing a boost vaccine for vaccinating a subjectthat has been vaccinated with a priming vaccine comprising a gBglycoprotein or its ectodomain 3-52 weeks, optionally 4-16 weeks prior,wherein the modified gB glycoprotein lacks the AD-1 epitope.

The phrase “lacks the AD-1 epitope” as used herein refers to a proteinor virus containing the gB protein wherein the AD-1 epitope is mutatedor absent. This allows expansion of the levels of relatively rare memoryB lymphocytes or naïve B cells that make high affinity antibodiesagainst the AD-2 epitope. In one embodiment, a fragment of gB that lacksthe AD-1 epitope but preserves AD-2 is used. Such a fragment may be madeup of the N-terminal ˜70-100 amino acids of the gB protein. Thisfragment does not include the amino acids that comprise the AD-1 epitope(Wagner et al 1992). It has been demonstrated that neutralizingantibodies that bind to the AD-2 epitope bind more tightly to thisN-terminal fragment of gB than they bind to the AD-2 linear peptide,indicating that in this fragment the AD-2 peptide adopts theconfiguration it does in the native gB (Thomson et al. 2008).

Also provided herein is a kit comprising a gB protein as disclosedherein and a modified gB protein as disclosed herein, wherein themodified gB glycoprotein lacks the AD-1 epitope.

In one embodiment the kit is used for inducing a cross-protectiveantibody response in a subject.

In an embodiment, the kit further comprises an instrument foradministering the gB proteins and/or instructions for use and/or acontainer.

Human Immune-Deficiency Virus (HIV)

It will be evident to those skilled in the art, that the presentlydisclosed methodology is also useful for eliciting broadly neutralizingantibodies against the envelope protein of HIV-1 gp160 or the ectodomaingp140 (Karlsson Hehestam et al 2008, and Kwong and Wilson 2009).

Accordingly, in yet another embodiment the present disclosure provides amethod of inducing a cross-protective neutralizing antibody response ina subject against HIV-1 comprising:

(a) administering a first unique gp140 or gp160 glycoprotein to thesubject; and

(b) administering a second unique gp140 or gp160 protein, 3-52 weeksafter a);

wherein the second unique gp140 or gp160 glycoprotein and the firstunique gp140 or gp160 glycoprotein are immunologically distinct butshare conserved sites that are not normally immunogenic for antibodies.

In another embodiment, the disclosure provides a use of a second uniquegp140 or gp160 glycoprotein for inducing a cross-protective antibodyresponse in a subject that has been previously subjected to immunizationwith a first unique gp140 or gp160 glycoprotein 3-52 weeks prior;wherein the second unique gp140 or gp160 glycoprotein and the firstunique gp140 or gp160 glycoprotein are immunologically distinct butshare conserved sites that are not normally immunogenic for antibodies.

The disclosure also provides second unique gp140 or gp160 glycoproteinfor use in inducing a cross-protective antibody response in a subjectthat has been previously subjected to immunization with a first uniquegp140 or gp160 glycoprotein 3-52 weeks prior; wherein the second uniquegp140 or gp160 glycoprotein and the first unique gp140 or gp160glycoprotein are immunologically distinct but share conserved sites thatare not normally immunogenic for antibodies.

In yet another embodiment, the disclosure provides a use of a secondunique gp140 or gp160 glycoprotein for preparing a boost vaccine forvaccinating a subject that has been vaccinated with a priming vaccinecomprising a first unique gp140 or gp160 glycoprotein 3-52 weeks prior,wherein the second unique gp140 or gp160 glycoprotein and the firstunique gp140 or gp160 glycoprotein are immunologically distinct butshare conserved sites that are not normally immunogenic for antibodies.

The term “unique gp140 or gp160 glycoprotein” as used herein refers to agp140 or gp160 glycoprotein or fragment thereof that is unique orunfamiliar to the subject it is desired to vaccinate, so that there areno or few memory B cells activated by contact with the “unique gp140 orgp160 glycoprotein” and there are no or low levels of antibodiescirculating in the subject that bind to the unique gp140 or gp160glycoprotein. In the “unique gp 140 or gp160 glycoprotein”, epitopesthat typically elicit a strong antibody response in the subject aredifferent, for example, due to mutation or deletion of amino acids ordifferent glycosylation, from epitopes on related gp140 or gp160glycoproteins previously encountered by the subject, whether by naturalinfection or vaccination. However, the “unique gp140 or gp160glycoprotein” shares common B-cell epitopes that are the target ofprotective antibodies in the subject but that are normally notimmunogenic because of competition for T cell help by the more numerousB cells stimulated by immunogenic parts of the pathogen antigen. The lowfrequency of memory B cells in the population to be vaccinated inducedby the unique glycoprotein that make antibodies that bind tightly to theputative unique protein can be readily ascertained using the methods ofWen et al 1987 or Corti et al 2010.

Also provided herein is a kit comprising a first unique gp140 or gp160glycoprotein as disclosed herein and a second gp140 or gp160glycoprotein as disclosed herein, wherein the first gp140 or gp160glycoprotein and the second gp140 or gp160 glycoprotein areimmunologically distinct but share conserved sites that are not normallyimmunogenic for antibodies.

In one embodiment the kit is used for inducing a cross-protectiveantibody response in a subject.

In an embodiment, the kit further comprises an instrument foradministering the gp140 or gp160 glycoproteins and/or instructions foruse and/or a container.

The term “subject” as used herein refers to any member of the animalkingdom, optionally humans. For example, a subject that is susceptibleto influenza infection, includes, without limitation, birds, pigs,horses and humans.

In an embodiment, the methods and/or uses described herein furthercomprise administration of an adjuvant with the first and/or secondpathogen antigen and/or third pathogen antigen or unique proteinsdisclosed herein. The term “adjuvant” as used herein refers to asubstance that is able to enhance the immunostimulatory effects of thepathogen antigens described herein but does not have any specificantigenic effect itself. Typical adjuvants include, without limitation,Freund's adjuvant, aluminium salts, squalene, poly I:C, GM-CSF, SB-AS2,Ribi adjuvant system, Gerbu adjuvant, CpG and monophosphoryl Lipid A andapproved proprietary adjuvants such as AS03 adjuvant system, an emulsioncomposed of DL-a-tocopherol, squalene and polysorbate 8 developed byGlaxoSmithKline (GSK).

The immunologically effective amount will, as a person of skill in theart will understand, vary with the formulation, the route ofadministration, the host being treated and the like but can neverthelessbe routinely determined by one skilled in the art.

The pathogen antigens or proteins disclosed herein in an embodiment aresuitably formulated as a liquid formulation, a solid formulation or aspray formulation.

In an embodiment, the pathogen antigens or proteins disclosed herein aresuitably formulated for oral, for example via drinking water and/orcombined with food; intranasal, for example via spray; eye drop;intramuscular; intradermal; subcutaneous; intravenous and/orintraperitoneal administration.

In one embodiment, the first and/or second pathogen antigen or proteinsdisclosed herein are administered subcutaneously, intramuscularly,intraperitoneally or intranasally. In an embodiment, the method ofadministration is the same for the first and second pathogen antigen orprotein disclosed herein. In an alternate embodiment, the method ofadministration is different for the first and second pathogen antigen orprotein disclosed herein.

Suitable carriers and/or pharmaceutically acceptable carriers includefor example water, including sterile water, saline, ethanol, ethyleneglycol, glycerol, water in oil emulsions, oil in water emulsions,saponins and alum based carriers etc and coformulants may be added.Pharmaceutically acceptable carriers include for example suitablecarriers that are suitable for animal administration, for example whichhave been filtered for sterility. Suitable vehicles are described, forexample, in Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa.,USA, 2000).

Assays for Detecting Cross-Protective Antibodies

In order to detect heterosubtypic antibodies that neutralize infectivityand are thus likely to be protective—for the purposes of assessingvaccination regimens or for screening for therapeutic or prophylacticmonoclonal antibodies, an assay must be sensitive to all inhibitoryantibodies, whether they neutralize by blocking binding of the virus tothe host cells or whether they block at a later step by blocking theconformational change in the HA stem that is necessary for the fusion ofthe membranes of the virus and the endosome and thus entry of the viralgenome into the cytosol.

The standard WHO microneutralization assay only allows the mixture ofthe antibodies under test and the challenge virus to be in contact withthe host cells for only a few hours after which the antibodies and virusare removed and then cells are incubated for 1-5 days to allow theinfection to develop. Thus, the antibodies have to act in the first fewhours of the infection. For conventional neutralizing antibodies thatbind to the HA head and block the attachment of the HA of the virus toits receptor on the host cell, this does not matter. However, forantibodies that block infectivity by binding to the HA stem and inhibitfusion of the virus to the endosomal membrane, they may have to bepresent in higher concentrations for longer time periods to blockinfectivity. Assays where antibodies are present for the entire 1-5 dayassay, allow antibodies to bind to the HA stem and neutralize, whilethey do not in the standard assay.

Accordingly, also provided herein is an assay for detectingcross-protective antibodies against an influenza virus comprising:

-   -   (a) incubating cells with the influenza virus and test serum        sample for 1-5 days; wherein the cells express a protease that        cleaves hemagglutinin of the influenza virus; and    -   (b) detecting viral infectivity compared to a control without        sample;    -   wherein a decrease in viral infectivity at 1-5 days indicates        the presence of cross-protective antibodies.

The term “viral infectivity” as used herein refers to the ability of thevirus to infect the host cells and can be evaluated by the amount ofviral proteins present or by the amount of cell survival. Thus,detecting viral infectivity includes, without limitation, detecting theamount of viral proteins and/or detecting cell survival.

In one embodiment, detecting viral infectivity comprises detecting cellsurvival and a decrease in viral infectivity comprises an increase incell survival.

The term “control” as used herein refers to a sample from a subject or agroup of subjects who have not been infected or vaccinated with novel orunique influenza. The term also includes a predetermined standard.

Cleavage of hemagglutinin is necessary for the hemagglutinin to undergothe conformational change that is necessary for fusion of the viralmembrane with that of the host cell. Some avian influenza viruses forexample are readily cleaved by enzymes prevalent in many cells. Howeverfor human and mammalian influenza viruses the enzymes that cleave thehemagglutinin are only present in a restricted set of cells whichinclude, without limitation, the cells that line the lungs. Thereforefor many years the assay that was used to detect neutralizing antibodiesthat are likely to be protective used mammalian cells (usually canineMDCK cells) and relied on including in the medium a proteolytic enzymesuch as TCPK-trypsin. For this reason, in the standard WHOmicroneutralization assay, the serum and virus mixture is incubated withthe MDCK cells for 2-3 hours and then removed lest it inhibitTCPK-trypsin.

Accordingly, in one embodiment, the cell line is a mammalian cell-linederived from the lung, airways or epithelial cells of the intestine. Forexample one human cell line that originates from the human airway isA549, which expresses proteolytic enzymes, which normally cleaveinfluenza hemagglutinin such as TMPRSS2 and HAT.

In another embodiment, the hemagglutinin of the influenza virus againstwhich it is desired to detect neutralizing antibodies against the stemmay be mutated at the cleavage site making it readily cleavableintracellularly like the hemagglutinin of avian influenza viruses.

Method for Generating Monoclonal Antibodies Cross-Protective AgainstInfluenza

The present demonstration that cross-reactive antibodies are producedare dominant in the response to an unfamiliar hemagglutinin allows forthe generation of monoclonal antibodies that cross-react with multipleinfluenza strains and/or subtypes. Accordingly also provided herein is amethod of generating monoclonal antibodies, optionally human,cross-protective against influenza comprising:

-   -   (a) isolating cells from a sample of blood or other tissue        containing cells of the immune system from a subject that has        been infected or vaccinated with an influenza strain that        exhibits a unique hemagglutinin;    -   (b) preparing monoclonal antibodies using the cells of (a); and    -   (c) selecting monoclonal antibodies that cross-react with        different strains and subtypes of viruses or that bind to the        stem of the hemagglutinin.

In one embodiment, the subject has been vaccinated using one of themethods or uses described herein for inducing a cross-protectiveantibody response to influenza.

The method for deriving immune cells that contain cells makingantibodies that cross-react with and neutralize many types of influenzaviruses, will be useful for the generation of monoclonal antibodiesproduced by a variety of methods well known to those skilled in the art,including display-based library techniques, immortalization or hybridomatechniques, or methods that rely on cloning or sequencing cDNA or RNAthat encode immunoglobulins from selected single cells that make thedesired antibodies. Mixtures of monoclonal antibodies cross-reactiveagainst multiple strains generated by this method may be combined tominimize the emergence of mutant strains that escape the neutralizationby these antibodies.

To produce human monoclonal antibodies, antibody producing cells(lymphocytes) can be harvested from a human infected with influenza andfused with myeloma cells by standard somatic cell fusion procedures thusimmortalizing these cells and yielding hybridoma cells. Such techniquesare well known in the art, (e.g. the hybridoma technique originallydeveloped by Kohler and Milstein (Nature 256:495-497 (1975)) as well asother techniques such as the human B-cell hybridoma technique (Kozbor etal., Immunol. Today 4:72 (1983)), the EBV-hybridoma technique to producehuman monoclonal antibodies (Cole et al., Methods Enzymol, 121:140-67(1986)) or EBV immortalization of activated B cells (Pinna et al. 2009),and screening of combinatorial antibody libraries (Huse et al., Science246:1275 (1989)). Monoclonal antibodies can be screened immunochemicallyfor production of antibodies specifically reactive with hemagglutininand the monoclonal antibodies can be isolated. Optional methods aredescribed in patent application PCT/CA 2006/001074.

Cross-Protective and/or Novel Monoclonal Antibodies to Influenza

The present inventor has obtained and sequenced ten human monoclonalantibodies cross-protective and/or novel to influenza using methodsdescribed in patent application PCT/CA2006/001074, incorporated hereinby reference. The human monoclonal antibodies were developed fromexposure of the human immune system to a unique hemagglutinin that wasimmunologically distinct from the HA of other influenza viruses thathumans in general had been exposed to. In the present case, the HA ofthe 2009 (H1N1) pandemic influenza virus of swine origin isantigenically unrelated to the hemagglutinin of any strain of influenzavirus to which most humans had been previously exposed to throughinfections or vaccinations, such that few humans had neutralizingantibodies against it (Itoh et al, 2009). The exposure to the unique HAmeant that there were very few memory B cells that made antibodies thatcross-react with the head of nH1N1 HA and thus could be activated by it.This circumstance allowed the relatively rare memory B cells thatrecognized regions of the HA that were conserved with seasonal influenzaviruses that the humans had been exposed to be activated by the HA ofthe nH1N1 and to access T-cell help and undergo affinity maturation.These memory B cells include those making antibodies against theconserved region of the stem and also some making antibodies againstconserved regions of the head of the HA.

The inventor has obtained ten monoclonal antibodies, V2-36, V2-7,V3-2G6, I8-1B6, V3-3D2, V3-1G10, I5-24, I4-128, V4-17 and V3-2C3, anddetermined the sequence of the light and heavy chain variable regionsand complementarity determining regions 1, 2 and 3 of the antibodies(Table 2).

Given the disclosure of the sequence of the heavy chain and light chainvariable region that determine the binding characteristics of a givenantibody, a person skilled in the art would readily be able to determinesequences of nucleotides that specify the synthesis of the antibody thatis disclosed.

The inventor has obtained the amino acid sequences of the variableregions of V2-36. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence YSNIGTGFD (SEQ ID NO:1); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence GNN (SEQ ID NO:2); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequenceQSFDSSLSGSNV (SEQ ID NO:3); isolated heavy chain CDR1 comprising theamino acid sequence GGSISGGSHY (SEQ ID NO:4); isolated heavy chain CDR2comprising the amino acid sequence IYYSGST (SEQ ID NO:5); and isolatedheavy chain CDR3 comprising the amino acid sequence AKHESDSSSWHTGWNWFDP(SEQ ID NO:6).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence YSNIGTGFD (SEQ ID NO:1); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence GNN (SEQ ID NO:2); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence QSFDSSLSGSNV (SEQ IDNO:3); the heavy chain CDR1 comprising the amino acid sequenceGGSISGGSHY (SEQ ID NO:4); the heavy chain CDR2 comprising the amino acidsequence IYYSGST (SEQ ID NO:5); and the heavy chain CDR3 comprising theamino acid sequence AKHESDSSSWHTGWNWFDP (SEQ ID NO:6).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:1, 2 and/or3), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:4, 5 and/or 6). Inone embodiment, the light chain variable region comprises the amino acidsequence shown in SEQ ID NO:7. In another embodiment, the heavy chainvariable region comprises the amino acid sequence shown in SEQ ID NO:8.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:7, and an isolated nucleic acid sequence encoding the heavychain variable region comprising the amino acid sequence shown in SEQ IDNO:8.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:1-3 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:4-6.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:1, 2 and 3 and/or the heavy chain CDRsequences of SEQ ID NOS:4, 5 and 6. In another embodiment, the antibodyor antibody fragment comprises the amino acid of SEQ ID NO: 7 (lightchain variable region) and/or the amino acid of SEQ ID NO:8 (heavy chainvariable region).

The inventor has obtained the amino acid sequences of the variableregions of antibody V2-7. Accordingly, the disclosure provides isolatedlight chain complementarity determining region 1 (CDR1) comprising theamino acid sequence STNIGAGLA (SEQ ID NO:9); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence GNT (SEQ ID NO:10); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequenceQSFDGSLSGSNV (SEQ ID NO:11); isolated heavy chain CDR1 comprising theamino acid sequence GGSIRGGTNY (SEQ ID NO:12); isolated heavy chain CDR2comprising the amino acid sequence VYYSGST (SEQ ID NO:13); and isolatedheavy chain CDR3 comprising the amino acid sequence ARHESDSSSWHTGWNWFDP(SEQ ID NO:14).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence STNIGAGLA (SEQ ID NO:9); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence GNT (SEQ ID NO:10); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence QSFDGSLSGSNV (SEQ IDNO:11); the heavy chain CDR1 comprising the amino acid sequenceGGSIRGGTNY (SEQ ID NO:12); the heavy chain CDR2 comprising the aminoacid sequence VYYSGST (SEQ ID NO:13); and the heavy chain CDR3comprising the amino acid sequence ARHESDSSSWHTGWNWFDP (SEQ ID NO:14).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:9, 10 and/or11), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:12, 13 and/or 14).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:15. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:16.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:15, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:16.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:9-11 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:12-14.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:9, 10 and 11 and/or the heavy chainCDR sequences of SEQ ID NOS:12, 13 and 14. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO: 15(light chain variable region) and/or the amino acid of SEQ ID NO:16(heavy chain variable region).

The inventor has obtained the amino acid sequences of the variableregions of V3-2G6. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence QIVSSSQ (SEQ ID NO:17); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence AAS (SEQ ID NO:18); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequence QQYGTSHA(SEQ ID NO:19); isolated heavy chain CDR1 comprising the amino acidsequence GGTFSSFA (SEQ ID NO:20); isolated heavy chain CDR2 comprisingthe amino acid sequence IIGMFGTT (SEQ ID NO:21); and isolated heavychain CDR3 comprising the amino acid sequence ARGKKYYHDTLDY (SEQ IDNO:22).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence QIVSSSQ (SEQ ID NO:17); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence AAS (SEQ ID NO:18); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence QQYGTSHA (SEQ IDNO:19); the heavy chain CDR1 comprising the amino acid sequence GGTFSSFA(SEQ ID NO:20); the heavy chain CDR2 comprising the amino acid sequenceIIGMFGTT (SEQ ID NO:21); and the heavy chain CDR3 comprising the aminoacid sequence ARGKKYYHDTLDY (SEQ ID NO:22).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:17, 18 and/or19), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:20, 21 and/or 22).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:23. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:24.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:23, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:24.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:17-19 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:20-22.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:17, 18 and 19 and/or the heavy chainCDR sequences of SEQ ID NOS:20, 21 and 22. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO: 23(light chain variable region) and/or the amino acid of SEQ ID NO:24(heavy chain variable region).

The inventor has obtained the amino acid sequences of the variableregions of I8-1B6. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence NSDVGTYNY (SEQ ID NO:25); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence DVS (SEQ ID NO:26); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequenceSSYTTSNTRV (SEQ ID NO:27); isolated heavy chain CDR1 comprising theamino acid sequence GGIFSNFA (SEQ ID NO:28); isolated heavy chain CDR2comprising the amino acid sequence ILSIFRTT (SEQ ID NO:29); and isolatedheavy chain CDR3 comprising the amino acid sequence ARSITNLYYYYMDV (SEQID NO:30).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence NSDVGTYNY (SEQ ID NO:25); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence DVS (SEQ ID NO:26); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence SSYTTSNTRV (SEQ IDNO:27); the heavy chain CDR1 comprising the amino acid sequence GGIFSNFA(SEQ ID NO:28); the heavy chain CDR2 comprising the amino acid sequenceILSIFRTT (SEQ ID NO:29); and the heavy chain CDR3 comprising the aminoacid sequence ARSITNLYYYYMDV (SEQ ID NO:30).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:25, 26 and/or27), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:28, 29 and/or 30).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:31. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:32.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:31, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:32.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:25-27 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:28-30.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:25, 26 and 27 and/or the heavy chainCDR sequences of SEQ ID NOS:28, 29 and 30. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO: 31(light chain variable region) and/or the amino acid of SEQ ID NO:32(heavy chain variable region).

The inventor has obtained the amino acid sequences of the variableregions of V3-3D2. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence QDISNY (SEQ ID NO:33); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence ATS (SEQ ID NO:34); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequence QQYSRYPPT(SEQ ID NO:35); isolated heavy chain CDR1 comprising the amino acidsequence GVIFNAYA (SEQ ID NO:36); isolated heavy chain CDR2 comprisingthe amino acid sequence ITGVFHTA (SEQ ID NO:37); and isolated heavychain CDR3 comprising the amino acid sequence ARGPKYYHSYMDV (SEQ IDNO:38).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence QDISNY (SEQ ID NO:33); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence ATS (SEQ ID NO:34); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence QQYSRYPPT (SEQ IDNO:35); the heavy chain CDR1 comprising the amino acid sequence GVIFNAYA(SEQ ID NO:36); the heavy chain CDR2 comprising the amino acid sequenceITGVFHTA (SEQ ID NO:37); and the heavy chain CDR3 comprising the aminoacid sequence ARGPKYYHSYMDV (SEQ ID NO:38).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:33, 34 and/or35), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:36, 37 and/or 38).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:39. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:40.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:39, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:40.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:33-35 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:36-38.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:33, 34 and 35 and/or the heavy chainCDR sequences of SEQ ID NOS:36, 37 and 38. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO:39(light chain variable region) and/or the amino acid of SEQ ID NO:40(heavy chain variable region).

The inventor has obtained the amino acid sequences of the variableregions of V3-1G10. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence QSVGTN (SEQ ID NO:41); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence GAS (SEQ ID NO:42); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequenceQHYNNWPPYT (SEQ ID NO:43); isolated heavy chain CDR1 comprising theamino acid sequence GVTFNHYT (SEQ ID NO:44); isolated heavy chain CDR2comprising the amino acid sequence IIPLFGTA (SEQ ID NO:45); and isolatedheavy chain CDR3 comprising the amino acid sequence ARSGTTKTRYNWFDP (SEQID NO:46).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence QSVGTN (SEQ ID NO:41); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence GAS (SEQ ID NO:42); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence QHYNNWPPYT (SEQ IDNO:43); the heavy chain CDR1 comprising the amino acid sequence GVTFNHYT(SEQ ID NO:44); the heavy chain CDR2 comprising the amino acid sequenceIIPLFGTA (SEQ ID NO:45); and the heavy chain CDR3 comprising the aminoacid sequence ARSGTTKTRYNWFDP (SEQ ID NO:46).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:41, 42 and/or43), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:44, 45 and/or 46).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:47. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:48.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:47, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:48.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:41-43 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:44-46.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:41, 42 and 43 and/or the heavy chainCDR sequences of SEQ ID NOS:44, 45 and 46. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO:47(light chain variable region) and/or the amino acid of SEQ ID NO:48(heavy chain variable region).

The inventor has obtained the amino acid sequences of the variableregions of I5-24. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence QSLSSGH (SEQ ID NO:49); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence GAS (SEQ ID NO:50); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequence QQYAVFLYT(SEQ ID NO:51); isolated heavy chain CDR1 comprising the amino acidsequence GGTFSRYT (SEQ ID NO:52); isolated heavy chain CDR2 comprisingthe amino acid sequence FIPLLGMT (SEQ ID NO:53); and isolated heavychain CDR3 comprising the amino acid sequence ARHDSSGYHPLDY (SEQ IDNO:54).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence QSLSSGH (SEQ ID NO:49); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence GAS (SEQ ID NO:50); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence QQYAVFLYT (SEQ IDNO:51); the heavy chain CDR1 comprising the amino acid sequence GGTFSRYT(SEQ ID NO:52); the heavy chain CDR2 comprising the amino acid sequenceFIPLLGMT (SEQ ID NO:53); and the heavy chain CDR3 comprising the aminoacid sequence ARHDSSGYHPLDY (SEQ ID NO:54).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:49, 50 and/or51), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:52, 53 and/or 54).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:55. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:56.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:55, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:56.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:49-51 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:52-54.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:49, 50 and 51 and/or the heavy chainCDR sequences of SEQ ID NOS:52, 53 and 54. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO:55(light chain variable region) and/or the amino acid of SEQ ID NO:56(heavy chain variable region).

The inventor has obtained the amino acid sequences of the variableregions of I4-128. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence QTISTY (SEQ ID NO:57); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence MAS (SEQ ID NO:58); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequence QHYNTYSST(SEQ ID NO:59); isolated heavy chain CDR1 comprising the amino acidsequence GGTFSTYG (SEQ ID NO:60); isolated heavy chain CDR2 comprisingthe amino acid sequence IIPIFGTA (SEQ ID NO:61); and isolated heavychain CDR3 comprising the amino acid sequence ARPNTYGYILPVY (SEQ IDNO:62).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence QTISTY (SEQ ID NO:57); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence MAS (SEQ ID NO:58); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence QHYNTYSST (SEQ IDNO:59); the heavy chain CDR1 comprising the amino acid sequence GGTFSTYG(SEQ ID NO:60); the heavy chain CDR2 comprising the amino acid sequenceIIPIFGTA (SEQ ID NO:61); and the heavy chain CDR3 comprising the aminoacid sequence ARPNTYGYILPVY (SEQ ID NO:62).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:57, 58 and/or59), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:60, 61 and/or 62).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:63. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:64.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:63, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:64.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:57-59 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:60-62.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:57, 58 and 59 and/or the heavy chainCDR sequences of SEQ ID NOS:60, 61 and 62. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO:63(light chain variable region) and/or the amino acid of SEQ ID NO:64(heavy chain variable region).

The inventor has obtained the amino acid sequences of the variableregions of V4-17. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence SSNIGTYY (SEQ ID NO:65); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence DNN (SEQ ID NO:66); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequenceAAWDDSLSGVV (SEQ ID NO:67); isolated heavy chain CDR1 comprising theamino acid sequence GGSITRNSYF (SEQ ID NO:68); isolated heavy chain CDR2comprising the amino acid sequence MYYDGTT (SEQ ID NO:69); and isolatedheavy chain CDR3 comprising the amino acid sequence ARHHVTELRVLEWLPKSDY(SEQ ID NO:70).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence SSNIGTYY (SEQ ID NO:65); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence DNN (SEQ ID NO:66); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence AAWDDSLSGVV (SEQ IDNO:67); the heavy chain CDR1 comprising the amino acid sequenceGGSITRNSYF (SEQ ID NO:68); the heavy chain CDR2 comprising the aminoacid sequence MYYDGTT (SEQ ID NO:69); and the heavy chain CDR3comprising the amino acid sequence ARHHVTELRVLEWLPKSDY (SEQ ID NO:70).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:65, 66 and/or67), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:68, 69 and/or 70).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:71. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:72.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:71, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:72.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:65-67 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:68-70.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:65, 66 and 67 and/or the heavy chainCDR sequences of SEQ ID NOS:68, 69 and 70. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO:71(light chain variable region) and/or the amino acid of SEQ ID NO:72(heavy chain variable region).

The inventor has obtained the amino acid sequences of the variableregions of V3-2C3. Accordingly, the disclosure provides isolated lightchain complementarity determining region 1 (CDR1) comprising the aminoacid sequence QSISSW (SEQ ID NO:73); isolated light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence KAS (SEQ ID NO:74); isolated light chain complementaritydetermining region 3 (CDR3) comprising the amino acid sequence QHYNSYSQT(SEQ ID NO:75); isolated heavy chain CDR1 comprising the amino acidsequence GGTFNNYA (SEQ ID NO:76); isolated heavy chain CDR2 comprisingthe amino acid sequence IIPIFGTA (SEQ ID NO:77); and isolated heavychain CDR3 comprising the amino acid sequence ARVCSFYGSGSYYNVFCY (SEQ IDNO:78).

The disclosure also includes isolated nucleic acid sequences encodingthe light chain complementarity determining region 1 (CDR1) comprisingthe amino acid sequence QSISSW (SEQ ID NO:73); the light chaincomplementarity determining region 2 (CDR2) comprising the amino acidsequence KAS (SEQ ID NO:74); the light chain complementarity determiningregion 3 (CDR3) comprising the amino acid sequence QHYNSYSQT (SEQ IDNO:75); the heavy chain CDR1 comprising the amino acid sequence GGTFNNYA(SEQ ID NO:76); the heavy chain CDR2 comprising the amino acid sequenceIIPIFGTA (SEQ ID NO:77); and the heavy chain CDR3 comprising the aminoacid sequence ARVCSFYGSGSYYNVFCY (SEQ ID NO:78).

Also provided are isolated light chain variable regions comprising lightchain CDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:73, 74 and/or75), and isolated heavy chain variable regions comprising heavy chainCDR1, CDR2 and/or CDR3 disclosed herein (SEQ ID NOS:76, 77 and/or 78).In one embodiment, the light chain variable region comprises the aminoacid sequence shown in SEQ ID NO:79. In another embodiment, the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:80.

The disclosure also includes an isolated nucleic acid sequence encodingthe light chain variable region comprising the amino acid sequence shownin SEQ ID NO:79, and an isolated nucleic acid sequence encoding theheavy chain variable region comprising the amino acid sequence shown inSEQ ID NO:80.

The disclosure further provides an antibody or antibody fragmentcomprising at least one light chain complementarity determining regionas shown in SEQ ID NOs:73-75 and/or at least one heavy chaincomplementarity determining region as shown in SEQ ID NOs:76-78.

In one embodiment, the antibody or antibody fragment comprises the lightchain CDR sequences of SEQ ID NOS:73, 74 and 75 and/or the heavy chainCDR sequences of SEQ ID NOS:76, 77 and 78. In another embodiment, theantibody or antibody fragment comprises the amino acid of SEQ ID NO:79(light chain variable region) and/or the amino acid of SEQ ID NO:80(heavy chain variable region).

The disclosure also provides variants of the CDR sequences, light chainand heavy chain variable sequences and antibodies comprising saidvariant sequences. Such variants include proteins that performsubstantially the same function as the specific proteins or fragmentsdisclosed herein in substantially the same way. For example, afunctional variant of a CDR or light chain or heavy chain variableregion or antibody will be able to bind to an antigen or epitoperecognized by the native CDR or light chain or heavy chain variableregion or antibody.

In one embodiment, the variant amino acid sequences of the light chainCDR1, CDR2 and CDR3, and the heavy chain CDR1, CDR2 and CDR3 have atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% sequence identity to the CDR sequences disclosed herein.

In another embodiment, the variant amino acid sequences of the lightchain variable region and the heavy chain variable region have at least50%, at least 60%, at least 70%, at least 80%, at least 90% or at least95% sequence identity to the light chain variable region and heavy chainvariable region sequences disclosed herein.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. An optional, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentdisclosure. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score-50, wordlength=3 to obtain aminoacid sequences homologous to a protein molecule of the presentdisclosure. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997, NucleicAcids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to performan iterated search, which detects distant relationships betweenmolecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g., ofXBLAST and NBLAST) can be used (see, e.g., the NCBI website). Anotheroptional, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

The disclosure also provides isolated nucleic acid sequences encodingvariants of the CDR sequences and variable region sequences discussedabove.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentdisclosure may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine.

The term “isolated nucleic acid sequences” as used herein refers to anucleic acid substantially free of cellular material or culture mediumwhen produced by recombinant DNA techniques, or chemical precursors, orother chemicals when chemically synthesized. An isolated nucleic acid isalso substantially free of sequences, which naturally flank the nucleicacid (i.e. sequences located at the 5′ and 3′ ends of the nucleic acid)from which the nucleic acid is derived. The term “nucleic acid” isintended to include DNA and RNA and can be either double stranded orsingle stranded, and represents the sense or antisense strand. Further,the term “nucleic acid” includes the complementary nucleic acidsequences.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified amino acids.

The term “isolated polypeptides” refers to a polypeptide substantiallyfree of cellular material or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized.

Variant nucleic acid sequences include nucleic acid sequences thathybridize to the nucleic acid sequences encoding the amino acidsequences disclosed herein under at least moderately stringenthybridization conditions, or have at least 50%, 60%, 70%, 80%, 90% or95% sequence identity to the nucleic acid sequences that encode theamino acid sequences disclosed herein.

The term “variant” as used herein includes modifications or chemicalequivalents of the amino acid and nucleic acid sequences disclosedherein that perform substantially the same function as the polypeptidesor nucleic acid molecules disclosed herein in substantially the sameway. For example, variants of polypeptides disclosed herein include,without limitation, conservative amino acid substitutions. Variants ofpolypeptides also include additions and deletions to the polypeptidesequences disclosed herein. In addition, variant sequences includeanalogs and derivatives thereof.

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, and chimeric antibodies. The antibodymay be from recombinant sources and/or produced in transgenic animals.The term “antibody fragment” as used herein is intended to includewithout limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers,minibodies, diabodies, and multimers thereof, multispecific antibodyfragments and Domain Antibodies. Antibodies can be fragmented usingconventional techniques. For example, F(ab′)2 fragments can be generatedby treating the antibody with pepsin. The resulting F(ab′)2 fragment canbe treated to reduce disulfide bridges to produce Fab′ fragments. Papaindigestion can lead to the formation of Fab fragments. Fab, Fab′ andF(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques.

The antibody or antibody fragments described herein also includefunctional variants of the sequences so that the antibody or antibodyfragment can bind to the HA protein.

In certain embodiments, the antibody or antibody fragment comprises allor a portion of a heavy chain constant region, such as an IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. In oneembodiment, the heavy chain constant region is an IgG1 heavy chainconstant region. Furthermore, the antibody or antibody fragment cancomprise all or a portion of a kappa light chain constant region or alambda light chain constant region. In one embodiment, the light chainconstant region is a kappa light chain constant region.

As described herein, to produce human monoclonal antibodies, antibodyproducing cells (lymphocytes) can be harvested from a human infectedwith influenza and then used to make monoclonal antibodies. For examplethey can be fused with myeloma cells by standard somatic cell fusionprocedures thus immortalizing these cells and yielding hybridoma cells.Such techniques are well known in the art, (e.g. the hybridoma techniqueoriginally developed by Kohler and Milstein (Nature 256:495-497 (1975))as well as other techniques such as the human B-cell hybridoma technique(Kozbor et al., Immunol. Today 4:72 (1983)), the EBV-hybridoma techniqueto produce human monoclonal antibodies (Cole et al., Methods Enzymol,121:140-67 (1986)), and screening of combinatorial antibody libraries(Huse et al., Science 246:1275 (1989)). Alternatively methods that copythe genes encoding the antibodies produced by individual B cells forexample the selected lymphocyte antibody method (Babcook et al 1996) maybe used. These methods can be used to screen for monoclonal antibodiesthat specifically react with hemagglutinin.

Specific antibodies, or antibody fragments, reactive againsthemagglutinin antigen or the stem region thereof may also be generatedby screening expression libraries encoding immunoglobulin genes, orportions thereof, expressed in bacteria with cell surface components.For example, complete Fab fragments, VH regions and FV regions can beexpressed in bacteria using phage expression libraries (See for exampleWard et al., Nature 341:544-546 (1989); Huse et al., Science246:1275-1281 (1989); and McCafferty et al., Nature 348:552-554 (1990)).

The term “light chain complementarity determining region” as used hereinrefers to regions of hypervariability within the light chain variableregion of an antibody molecule. Light chain variable regions have threecomplementarity determining regions termed light chain complementaritydetermining region 1, light chain complementarity determining region 2and light chain complementarity determining region 3 from the aminoterminus to the carboxy terminus.

The term “light chain variable region” as used herein refers to thevariable region of a light chain.

The term “heavy chain complementarity determining region” as used hereinrefers to regions of hypervariability within the heavy chain variableregion of an antibody molecule. The heavy chain variable region hasthree complementarity determining regions termed heavy chaincomplementarity determining region 1, heavy chain complementaritydetermining region 2 and heavy chain complementarity determining region3 from the amino terminus to carboxy terminus.

The term “heavy chain variable region” as used herein refers to thevariable region of a heavy chain.

The disclosure also provides compositions comprising the CDRs in anappropriate framework, variable regions and/or antibodies disclosedherein with a pharmaceutically acceptable excipient, carrier, buffer orstabilizer.

Further provided herein are methods and uses of the CDRs in anappropriate framework, variable regions and/or antibodies or antibodyfragments thereof disclosed herein for protecting against infection withinfluenza or treating an infection with influenza in a subject. In oneembodiment, the disclosure provides a method for protecting againstinfection with influenza or treating an infection with influenza in asubject comprising administration of the CDRs in an appropriateframework, variable regions and/or antibodies or antibody fragmentsdescribed herein to a subject. Also provided is use of the CDRs in anappropriate framework, variable regions and/or antibodies or antibodyfragments described herein for protecting against infection withinfluenza or treating an infection with influenza in a subject. Furtherprovided is use of the CDRs in an appropriate framework, variableregions and/or antibodies or antibody fragments described herein forpreparing a medicament for protecting against infection with influenzaor treating an infection with influenza in a subject. Even furtherprovided are the CDRs in an appropriate framework, variable regionsand/or antibodies or antibody fragments described herein for use inprotecting against infection with influenza or treating an infectionwith influenza in a subject.

The above disclosure generally describes the present disclosure. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of thedisclosure. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES

Three approaches were used to generate human monoclonal antibodies(mAbs) that reacted with HA of the soluble ectodomain of the nH1N1influenza virus (snH1 HA) (FIG. 1). First, antibodies were randomlycloned from newly generated plasmablasts (PB) circulating in the bloodof recently infected patients. This approach was based upon observationsthat ˜7 days after vaccination, PB that secrete antibodies specific forthe vaccine appear in the blood (Barington et al. 1990; Heilmann et al.1987) and form a significant fraction of the total PB (Odendahl et al.2005; Wrammert et al. 2008), and upon techniques that the presentinventor had previously used to obtain monoclonal antibodies fromblood-borne PB by RT-PCR and cloning and expression of the DNA encodingthe antigen-binding site (Babcook et al. 1996). In the absence of dataon the kinetics of entry of infection-specific PB into the blood duringinfections, blood was collected from subjects with laboratory-confirmednH1N1 infections ˜7 days after the onset of symptoms, andfluorescence-activated cell-sorting (FACS) was used to purify individualPB. From these, 8 mAbs were cloned and expressed as IgG1 molecules(Babcook et al. 1996; McLean et al. 2005), finding 4 that bound to arecombinant soluble form of the trimeric ectodomain of the nH1N1 HA(snH1 HA), (Table 1a, and the example of mAb I4-128 in FIG. 2).Strikingly, given the novel or unique antigenic nature of the nH1 HA,all 4 of these antibodies also bound to the current seasonal influenzavaccine (Table 1a, and example of mAb I4-128 in FIG. 2A). In the secondapproach, using similar blood samples, individual PB expressingantibodies were FACS-purified by using fluorochrome-labelled snH1 HA byexploiting the fact that newly generated PB express their immunoglobulinon their surface (Odendahl et al. 2005; Nossal et al. 1972). Five mAbswere generated that bound to snH1 HA and all 3 also bound to theseasonal influenza vaccine. Thus a total of 9 mAbs against snH1 HA weregenerated from blood-borne PB from 3 patients (Table 1a), demonstratingthat, during an infection, at least some newly generated PB enter theblood and do not all remain in lymphoid tissues near the site ofinfection. Sixteen more mAbs were also generated from snH1 HA-binding PBfrom subjects vaccinated with the nH1N1 vaccine. Strikingly, of thetotal of 25 mAbs generated from PB from infected or vaccinated subjects,all but one, V4-17, cross-reacted with the seasonal influenza vaccineand purified recombinant H5 HA and thus were also cross-reactive orheterosubtypic (Table 1a; FIG. 2A). In a third approach, blood sampleswere collected 2-8 weeks after recovery from infection or vaccinationwith pandemic 2009 (H1N1) influenza and FACS was used to purifyindividual snH1 HA-binding, class-switched memory B cells that were thenexpanded and differentiated to clones of PB as before (McLean et al.2005). Of the 23 mAbs against snH1 HA generated from the memory B cellsin this way, all cross-reacted with seasonal influenza vaccine andpurified recombinant H5 HA and were thus heterosubtypic (Table 1B;representative mAbs in FIGS. 2A and 2E, F).

In total, from 5 infected and 3 vaccinated subjects, 48 recombinant mAbswere generated that bound snH1 HA, and all but one cross-reacted withthe current seasonal influenza vaccine and purified recombinant H5 HA(Table 1). The high frequency of heterosubtypic mAbs was not due to thefact that recombinant snH1 HA was used to sort out PB or memory B cells,as all 4 of the snH1 HA-binding mAbs generated from the randomlyselected PB also bound to the seasonal influenza vaccine and thepurified recombinant H5 HA (Table 1A). Moreover, the epitopes bound bythe mAbs were present on the inactivated, detergent-disrupted nH1N1virus as all 48 mAbs also bound to the nH1N1 vaccine and 47 also boundto the 2009/2010 seasonal influenza vaccine and the purified recombinantH5 HA (Table 1; examples in FIGS. 2A and 2E, F).

Strikingly, 52% of the anti-snH1 HA mAbs, derived from infected (44%) orvaccinated subjects (57%), or PB (40%) or memory B cells (65%), used oneV-gene, IGHV1-69 (Table 1; FIG. 2B). IGHV1-69 is normally expressed 3.6%to less than 5% of B cells (de Wildt et al, 1999; Sasso et al. 1996).Where multiple mAbs were obtained from the same subject, the response toinfection or vaccination with nH1N1 was clonally diverse, although V4had two mAbs against the HA stem that were derived from the same B cellclonotype. The striking exception was one subject V2 (Table 1) which didnot yield a monoclonal antibody using IGHV1-69, discussed below. Insubject V3, 12 out of 14 mAbs derived from memory B cells used IGHV1-69,but these were all different with different IGHD and IGHJ genes and useda variety of L-chain V-genes. IGHV1-69 was used preferentially inrecombinant single-chain variable fragments (scFv) selected fromphage-display libraries of human immunoglobulin genes using the HA ofH5N1 avian influenza (Ekiert et al. 2009; Sui et al. 2009; Throsby etal. 2008; Kashyap et al. 2008). IGHV1-69 encodes major features of abinding site for a site on the HA stem that is highly conserved in avariety of influenza subtypes (Ekiert et al. 2009; Sui et al. 2009).This conservation reflects structural constraints on the HA stem, whichundergoes a pH-induced, irreversible conformational change when thevirus is endocytosed and encounters the low pH in the endosome (˜pH5)(Wiley et al. 1987). This conformation of the HA stem mediates fusion ofthe viral and endosomal membranes enabling the viral genome to enter thecytosol. Binding of these scFv prevented this conformational change inthe HA stem and results in neutralization of infectivity of multiplesubtypes of influenza (Ekiert et al. 2009; Sui et al. 2009). This raisedthe prospect that similar antibodies might be induced by a vaccine thatwould protect against multiple influenza subtypes.

The present data show that antibodies that bind to the HA of multiplestrains or subtypes of influenza viruses, including those usingIGHV1-69, can be readily made by humans in response to infection orvaccination by the nH1N1 influenza, which is a unique hemagglutinin tomost humans, and can dominate the antibody response. In ongoing work onanother subject who was vaccinated with non-adjuvanted nH1N1 vaccine,the first mAb from an snH1 HA-binding PB was generated and it wascross-reactive/heterosubtypic and used IGHV1-69.

The majority of the heterosubtypic mAbs bound to the HA stem but othersbound to the HA head. To test whether the epitope was on the HA stemversus the head, 3 approaches were used. To test whether the epitopebound by a mAb was on the HA stem two assays were used. One wasinhibition of binding to HA of C179, an antibody that was known to bindto a conserved epitope on the HA stem (Okuno et al. 1993). The secondassay was whether the epitope targeted by the mAb was affected bytreatment at low pH5, which induces an irreversible conformationalchange in the stem (Wiley et al. 1987). To test whether the epitope wason the HA head, close to the receptor-binding site, it was testedwhether the mAbs could inhibit hemagglutination by nH1N1. That mAbsusing IGHV1-69 bound to an epitope on the HA stem was confirmed byshowing that they inhibited completely binding of C179 to nH1N1 vaccine(FIG. 2C, upper graph). Another heterosubtypic mAb I5-52, which usedIGHV1-18, also inhibited C179-binding and thus bound to the HA stem(FIG. 2C). Additionally, low pH-treatment decreased the binding of mAbsusing IGHV1-69 selectively as compared with another heterosubtypic mAbV2-36 that used IGHV4-39 (FIG. 2C, lower graph). The IGHV1-69-using mAbsdid not inhibit hemagglutination by the nH1N1 virus (FIG. 2D),consistent with these mAbs binding to the stem rather than the HA head.In contrast, V2-36, which used IGHV4-39 and V4-12 inhibitedhemagglutination by nH1N1 (FIG. 2D), indicating that they bound to thehead of the nH1N1 HA and not the stem. All of these mAbs against the HAstem, both the 52% using IGHV1-69 and 6% using other IGHV-genes allbound to the conventionally produced pandemic H1N1 vaccine and to the2009/2010 seasonal influenza vaccine (FIG. 2A and Table 1). Thusconventional influenza vaccines exhibited the conserved epitope on theHA stem to which these heterosubtypic anti-HA stem antibodies bound. Itwas concluded from these data that the conserved epitope on the HA stemwas exhibited by the conventionally prepared influenza vaccines as wellas the epitopes on the HA head recognized by mAbs that inhibitedhemagglutination such as V2-36. This taught against the prevailing viewthat the conserved epitope on the HA stem was somehow hidden ininfectious influenza virus or vaccines (Chen et al, 2009, Corti et al,2010, Sui et al 2009, Steel et al 2010).

The present inventor obtained 9 mAbs that used IGHV4-39, and allinhibited hemagglutination, suggesting they bound an epitope on the HAhead. Eight of them were obtained from one subject, V2, who exhibited anoligoclonal response with 7 belonging to the same clonotype (comprisingV2-36, V2-2, V2-3, V2-4, V2-7, V2-11 and V2-38). Another mAb from thisdonor, V2-12 also used IGHV4-39 but combined it with different IGHD andIGHJ genes and a different L-chain V-gene. IGHV4-39—using mAbs dominatedthe response in this donor as the only other mAb obtained from thisdonor used another IGHV gene. Another mAb that used IGHV4-39, V4-17, wasobtained from another donor, V4, and bound to the HA head, as itinhibited hemagglutination and failed to inhibit the binding of C179(FIG. 2C). Although it did not bind to the 2009/2010 seasonal influenzavaccine, as discussed below, the evidence that V4-17 had 19 mutations inIGHV4-39 indicated that the PB was generated by the HA from nH1N1stimulating a memory B cell that had previously been induced byinfection or vaccination with a seasonal influenza H1N1 virus (thatdiffered in that epitope to the H1N1 influenza virus in the 2009/2010seasonal influenza vaccine). Thus it is likely that V4-17 is alsocross-reactive and will have reactivity with a previously encounteredseasonal influenza H1N1 virus.

Given that IGHV1-69 using mAbs had been shown to bind to the HA of H5N1viruses, the ability of some of the mAbs to bind to cells expressing thefull-length H5 HA from the highly pathogenic avian influenza A HongKong156/97 (H5N1) were tested (FIGS. 2E,F). IGHV1-69-using mAbs (FIG. 2E,F), as well as other heterosubtypic mAbs using other IGHV genes such asV3-1E8 and I5-52 bound the H5 HA. In contrast, V2-36 and V4-17, whichrecognize epitopes on the nH1 HA head, bound only weakly to the H5 HA ofHongKong 156/97 (H5N1) (FIG. 2F). Significant levels of antibodies thatbound to cells expressing H5 HA were also found in convalescent plasmafrom nH1N1 infected patients and in vaccinated subjects 7 days aftervaccination (FIG. 2F). All mAbs, with the sole exception of V4-17 werereactive in ELISA with purified recombinant H5 HA (A/Vietnam/1203/2004,Clade1) (FIG. 2A), Table 1.

32 of the mAbs were tested for their capacity to neutralize nH1N1infectivity in the standard WHO microneutralization assay. Only two ofthe mAbs that were tested (V2-36 and V4-17) neutralized infectivity ofnH1N1. Only V2-36 neutralized the seasonal influenza virusA/Brisbane/59/07(H1N1), consistent with the lack of binding of V4-17 tothe seasonal influenza vaccine (FIG. 2A). V2-36 and V4-17 were the onlymAbs of the 32 tested that inhibited hemagglutination. It was reasonedthat the standard assay was biased to mAbs that bound to epitopes on theHA head because they inhibited viral attachment. Thus in the standardmicroneutralization assay, mixtures of mAbs and the challenge virus wereonly in contact with the cells for 2-3 hours, after which they removed.This was because the assay was designed for assessing neutralizingantibodies in human serum and the removal of the serum (but not thepurified mAbs) was necessary to allow the TPCK-trypsin included in themedium to activate the virus by cleaving the HA (Klenk et al. 1975). AsmAbs that bind to the HA stem will not inhibit viral attachment and arewashed away after the 3 hour incubation with the antibody-virus mixture,the assay was modified by leaving the virus and mAbs for the duration ofthe assay. Under these conditions, which mimic the constant presence ofantibodies in an infection in vivo, most of the mAbs that bound to theHA stem exhibited neutralizing activity against the nH1N1 virus (FIG.3A). All of the 10 IGHV1-69-using mAbs tested in the modified assaycompletely neutralized nH1N1 at concentrations from 78-2500 ng/ml. Manyneutralized seasonal influenza A/Brisbane/59/07(H1N1). V3-1B9 usingIGHV3-11, and I14-2B7 using IGHV1-18, also completely neutralized nH1N1at 625 ng/ml and 1250 ng/ml respectively. However, in this panel ofmAbs, the most potent neutralizer was V2-36, which bound to the HA headand inhibited infectivity completely at less than 40 ng/ml. The secondwas V3-2G6 which neutralized completely at less than 80 ng/ml.

The ability of selected mAbs to neutralize the infectivity of the highlypathogenic avian influenza virus A/Goose/Ger/R1400/07 (H5N1) were testedand a good correlation was seen between H5 HA-binding (FIG. 2F) andneutralization of H5N1 infectivity (FIG. 3B), with V2-36 failing toneutralize H5N1 infectivity. As expected (Sui et al. 2009; Throsby etal. 2008), these IGHV1-69-using mAbs did not neutralize infectivity ofan H7N7 virus (FIG. 4). Moreover, plasma from subjects infected (I14) orvaccinated (V2) with nH1N1 also neutralized H5N1 infectivity (FIG. 3B).These mAbs inhibited cell-cell fusion mediated by cell-surfaceexpression of full-length HA from influenza A/Hong Kong/156/97 (H5N1)(Sui et al. 2009) (FIG. 3C). Thus it was concluded that humans infectedor vaccinated with nH1N1 make cross-protective antibodies that can bindto the HA of different virus subtypes and can neutralize pandemic nH1N1,seasonal H1N1, and highly pathogenic H5N1 avian influenza viruses.

Did the nH1 HA induce the production of these heterosubtypic mAbs byactivating a naïve B cell or a memory B cell generated in a previousencounter with influenza viruses? Especially in the case of the mAbsgenerated from PB collected ˜7-10 days after infection or vaccination(e.g. I5-24, V4-29), a large number of somatic mutations would suggestthat the nH1 HA cross-activated a pre-existing memory B cell. In fact,an unprecedented 52% of the mAbs generated from PB had IGHV genes withmore than 28 mutations, meaning that more than 10% of their nucleotideswere mutated. The median number of mutations in mAbs from PB was 29.This compares with the average mutation rate of human germinal centre ormemory B cells of 13.6+/−4.8 (Wrammert et al. 2008). Overall, theanti-snH1 HA mAbs from PB had a significantly higher frequency ofmutations in IGHV gene than those from memory B cells (median number ofmutations of 15.5) (FIG. 5A) and none of the memory B cells had morethan 10% of the nucleotides in IGHV mutated. This is consistent withevidence that murine PB make higher affinity antibodies than do memory Bcells (Smith et al. 1997). Based on the present findings it waspredicted that high-affinity memory B cells do not circulate in theblood until after retained antigen has been cleared from lymph-nodesbecause this antigen will activate them to PB.

It is likely that those cross-reactive or heterosubtypic antibodies thathad accumulated a lot of somatic mutations in the IGHV gene had beengenerated by activation by the HA of nH1N1 of memory B cells that hadbeen induced by previous contact with seasonal influenza viruses. Thisnotion is supported by a paper showing that vaccination with seasonalinfluenza expanded in some subjects a small population of memory B cellsmaking heterosubtypic antibodies that used IGHV1-69 (Corti et al. 2010).However, in contrast to the response to a unique HA, as in the infectionor vaccination with nH1N1 disclosed herein, the heterosubtypic responsereported in Corti et al (2010) was weak and only seen in someindividuals. Indeed as commented by Corti et al, their observationsraise the question of the effectiveness of heterosubtypic antibodiesinduced by influenza vaccinations as “even in high-responderindividuals, heterosubtypic antibodies hardly reach effectiveneutralizing concentrations in the serum”. Consistent with the inductionof low levels of heterosubtypic antibodies by seasonal influenza, a lowpercentage of European sera (Garcia et al. 2009) or pooled human gammaglobulin preparations contain low levels of antibodies against the H5N1HA (Lynch et al 2009). Human mAbs generated before the 2009 pandemicthat bind to the HA stem can neutralize nH1N1 (Burioni et al 2010) andvaccination with seasonal influenza induces increases in neutralizingtitres against H5N1 (Gioia et al. 2008), and the numbers of memory Bcells making antibodies that bind H5 HA (Corti et al. 2010). Moreover inone subject that was vaccinated with nH1N1, before vaccination therewere existing levels of antibodies binding to H5 HA-expressing cellsthat had increased by 7 days after vaccination (FIG. 5B). A minority ofthe mAbs that exhibited small numbers of somatic mutations, wereprobably ultimately derived from naïve B cells that were activated bynH1N1, whether directly generated from PB (I4-112, I5-52, or I4-128 with5, 11 or 13 mutations) or memory B cells (e.g. I4-1G8, I14-B7, V3-3B3,V3-2C3, or V3-2C2 with 3, 5, 6, 6 and 9 mutations). These mAbs suggest,without wishing to be bound by any theory, that the initial primaryresponse to an unfamiliar antigenically distinct unique influenzahemagglutinin is cross-reactive or heterosubtypic. Given that the memoryresponse to seasonal influenza is dominated by a limited number ofclonotypes of subtype-specific antibodies (Wrammert et al. 2008), withsubsequent encounters with slightly mutated hemagglutinins (“antigenicdrift”) these initial cross-reactive or heterosubtypic memory B cellsare rapidly outcompeted by higher affinity strain-specific memory Bcells generated by affinity maturation.

The present inventor tested the therapeutic potential of these mAbs intreating serious infections with nH1N1 in mice given a lethal dose of ahuman isolate of nH1N1 virus. The mAb V2-36 was given 24 hours afterinfection of the mice. Whereas 5/5 mice in the control group treatedwith only saline died, all 5 of the mice treated with a single injectionof 180 μg (˜6 mg/kg) of V2-36 24 hours after infection of the mice,survived (FIG. 6). A single dose of 200 μg of V2-36 given 48 hours afterinfection still had a therapeutic effect (FIG. 7). Also shown in FIG. 7are the therapeutic effects on mice with a lethal influenza infectionwith 300 μg of V3-2G6, I5-24, I4-128, V3-3D2, V3-1G10 and I8-1B6 and amixture of V2-36 and I5-24.

To test whether a heterosubtypic mAb against the HA stem generated froma vaccinated subject, V3-2G6, had therapeutic effects on a lethalinfection with avian H5N1 influenza, groups of mice were infected with aheterologous H5N1 influenza virus and given a single intraperitonealinjection of 150 μg or 300 μg of V3-2G6 24 hours after intra-nasalinfection (FIG. 8A). A 3^(rd) and 4^(th) group of mice that were treatedwith V3-2G6 were given single doses of 300 μg or 600 μg 48 hours afterinfection (FIG. 8A). There was 100% survival in all treatment groups,although the mice treated 48 hrs later were seriously ill, as evidencedby their loss of weight. To test the minimum dose that was necessary fora cross-protective therapeutic effect on lethal H5N1 influenzainfections, groups of mice were infected with a heterologous H5N1influenza virus and given a single intraperitoneal injection of 150 μgor 75 μg or 37.5 μg of V3-2G6 24 hours after intra-nasal infection (FIG.8B). As can be seen 37.5 μg of V3-2G6 only cured 80% of mice from alethal infection with H5N1 influenza virus, but the mice treated with 75ug, although they all survived had a larger weight loss compared withthe group treated with 150 μg.

To test whether a heterosubtypic mAb against the HA stem generated froma vaccinated subject, V3-2G6, protected mice from a lethal dose of aheterologous H5N1 influenza virus, groups of mice were treated withgraded doses of V3-2G6 and 24 hours later the mice were infectedintranasally. V3-2G6 was very potent. Both doses, 15 μg and 5 μg (250μg/Kg and 750 μg/Kg) protected against death from infection with H5N1,and 750 μg/Kg protected from any weight loss (FIG. 9).

It was next asked whether the dominant cross-protective antibodyresponse in memory B cells induced by vaccination with the pdmH1N1vaccine correlated with circulating cross-protective antibodies in humanplasma. The ability of plasma from donor V3 taken 14 days and one yearafter vaccination to protect mice against a lethal infection was testedwith H5N1 influenza (FIG. 10). As a control, plasma from a young adultdonor taken in 2006 was used, to ensure the subject could not have beenin contact with the 2009 pandemic H1N1 virus. It was observed that 400μl of plasma from V3 taken 14 days after vaccination completelyprotected the mice from a lethal infection with H5N1 influenza. 400 μlof plasma collected a year after the vaccination protected against deathbut there was a small weight loss with H5N1 infection. However threetimes the dose of human plasma protected the mice against significantweight loss (FIG. 10). These data showed that, even a year aftervaccination, human plasma conferred protection on mice subsequentlyinfected with the H5N1 influenza virus.

Why should cross-protective antibodies dominate the response to HA of apandemic influenza but not to seasonal influenza (Corti et al 2010)? Inthe response to a pandemic influenza virus, the only memory B cells thatbind the HA with sufficient affinity to differentiate them to a PB orinduce them to undergo somatic mutation in a germinal centre, will bethose making heterosubtypic antibodies that bind to conserved structuralfeatures among viral subtypes. Notably, helper T cells are activated byepitopes shared between viral subtypes (Doherty and Kelso, 2008) and Bcells that have been activated by binding HA or viruses will compete topresent these epitopes to the T cells (Allen et al. 2007). In thisregard, the memory B cells activated by conserved epitopes on the nH1 HAwill have multiple competitive advantages over the naïve B cells thatrecognize the novel or unique strain-specific features on the head ofthe pandemic HA. Not only are the heterosubtypic memory B cells morenumerous, they possess intrinsic advantages, including increasedsignalling and expression of proteins that co-activate T cells (Tangyeet al. 2009). Thus in the response to pandemic influenza, heterosubtypicantibodies will dominate. In contrast, with seasonal influenza, largenumbers of memory B cells against strain-specific epitopes on the HAhead are activated by low-affinity interactions with the HA of “drifted”seasonal strains and enter germinal centres to undergo affinitymaturation (Paus et al. 2006). There they out-compete the less numerousheterosubtypic memory B cells for T-cell help, explaining the rarity andlimited level of the heterosubtypic antibody responses to the HA ofseasonal influenza (Corti et al. 2010; Garcia et al. 2009).

In only one of the subjects, (V2), from whom 9 mAbs were generated thatbound to snH1 HA, did the present inventor fail to find a mAb usingIGHV1-69. Clearly B cells from V2 did express IGVH1-69 as otherantibodies were cloned using IGVH1-69 in V2. To obtain insight into themechanism of the dominant anti-HA stem antibody response to the pdmH1N1,the exceptional subject, V2, was analysed in more detail. If a frequencyof anti-HA stem mAbs using IGHV1-69 in responses to pdmH1N1 was assumedto be 50%, the probability of obtaining the observed result from V2 bychance alone was 0.002 and thus unlikely (Table 3). Similarities werenoted in the antibody response of V2 to the nH1N1 vaccine to that of thetypical human antibody response to the seasonal influenza vaccine. Thus8 of 9 mAbs from V2 blocked hemagglutination and were directed againstthe HA head and 7 belonged to the same clonotype (Table 1). Moreoverthese mAbs had many mutations and reacted with seasonal influenzavaccine, indicating they were derived from memory B cells that had beencross-activated by the HA of nH1N1 vaccine. Thus, in V2, the lack of adominant response of antibodies using IGHV1-69 to the nH1N1 vaccinecorrelated with an antibody response against the HA head, like thatwhich occurs in seasonal influenza (Wrammert et al, 2008).

In this subject, V2, aged 63, cross-reactive memory B cells usingIGHV4-39, were likely elicited by a virus related to nH1N1 (Hancock etal. 2009). Consistent with the notion that cross-reactive antibodiesagainst the HA head of seasonal influenza use IGHV4-39, a heterosubtypicantibody that cross-reacted with the head of some seasonal influenzaH1N1 and H5N1 viruses and that also used IGHV4-39, was recently isolatedfrom a subject vaccinated with the seasonal influenza vaccine (Corti etal. 2010). These activated memory B cells against the HA head, as inseasonal influenza, would have physically outcompeted (Schwickert et al,2011) for T cell help (Allen et al, 2007, Victora et al 2010, Schwickertet al 2011) those rare memory B cells against the HA stem. V2 was over60 years in age and clearly by his dominant, cross-reactive antibodyresponse to the HA head, with many somatic mutations (Table 1), had beenin contact with a related virus, as supported by Hancock et al (2009).In contrast, in other subjects infected or vaccinated with pdmH1N1, whosaw the HA head of nH1N1 as unique, there was a paucity of memory Bcells activated by the HA head of nH1N1. In them, those rare memory Bcells against the HA stem that had endocytosed HA of nH1N1 or nH1N1virions (Russell et al, 1979), could present T-cell epitopes sharedbetween viral subtypes (Doherty et al, 2008) to memory helper T cells,unimpeded by competition from memory B cells against the HA head (Allenet al, 2007, Victora, et al 2010, Schwickert et al 2011). Even if a fewmemory B cells had low affinity for the HA of nH1N1, they would notcompete for T cell help with memory B cells making higher-affinityantibodies against the HA stem (Victora et al 2010, Schwickert et al2011). Moreover, memory B cells against the HA stem would have multiplecompetitive advantages over the naïve B cells (Tangye et al 2009).Therefore antibodies against the HA stem will dominate the antibodyresponse to a novel HA. Moreover, it is unlikely that that the intrinsicnature of the pdmH1N1 antigen or its presentation to the immune system(Wei et al, 2010b) was a factor in the dominance of the anti-HA stemantibody response to pdmH1N1, as this occurred in our studies withsubjects with both infections and vaccinations with nH1N1, which involvemany differences in the presentation and nature of the antigen, the siteof the immune response, and the associated inflammatory responses.

Discussion

The present findings have implications for the rapid preparation oftherapeutic agents for emerging pandemics and for the prospect of abroad-spectrum influenza vaccine. In August 2009, only months after thenH1N1 outbreak had started, the present inventor obtained the firsttherapeutic mAb against nH1N1 (14-128). Furthermore, in principle, thereare no technical or regulatory reasons that therapeutic monoclonalantibodies generated in humans against pandemic influenza virus or otheremerging pathogens should not be rapidly developed and deployed.

The conventionally prepared nH1N1 vaccine as well as the seasonalinfluenza vaccine clearly exhibited the conserved epitope recognized bymAbs against the HA stem, including those mAbs that use IGHV1-69 (FIG.2A). The anti-HA stem mAbs bound as readily to the conventionallyprepared vaccines as did the mAbs that bound to the head and inhibitedhemagglutination like V2-36 (FIG. 2A). The nH1N1 vaccine wasadministered with the AS03 adjuvant system. However, two mAbs weregenerated against HA of nH1N1 from a subject vaccinated withunadjuvanted nH1N1 vaccine and one of the mAbs used IGHV1-69. Thus it islikely that unadjuvanted nH1N1 vaccine induces a high frequency ofanti-HA stem antibodies. Human vaccination with the nH1N1 vaccine,comprising a unique influenza HA to most humans induced circulatingheterosubtypic antibodies that, when transferred to mice, protectedagainst a lethal infection with heterologous H5N1 influenza virus (FIG.10). The present observation of a dominant heterosubtypic antibodyresponse to vaccination with a conventionally prepared pandemic nH1N1influenza supports a novel, vaccination strategy that deliberatelyavoids inducing antibodies against the HA head that normally protectagainst influenza infections. For example, the first vaccination couldbe with an inactivated influenza virus (or its HA) that has anantigenically unique HA head to that subject, such as that of thepandemic nH1N1. After an interval, this could be followed by vaccinationwith an inactivated influenza virus (or the HA) that shares a conservedepitope on the HA stem but has another antigenically unique HA head,such as that of a influenza virus circulating in another species likeavian influenza H5N1 virus or an artificially mutated HA. By avoidingthe cross-activation of memory B cells making antibodies with low orhigh affinity binding to epitopes on the HA head that will otherwiseoutcompete the rare heterosubtypic memory B cells making antibodiesagainst the conserved HA stem, this strategy ensures a robustcross-protective heterosubtypic antibody response. This is achievedbased on these results with pandemic nH1N1 vaccine, using conventionalvaccines based on HA from influenza viruses that have not circulated inhumans. One example is the existing approved vaccine for avian influenzaH5N1 virus. Boosting should be done with yet another unique HA head, forexample the avian H7 HA in the group 2 of subtypes of HA, in order toextend the range of broadly protective antibodies to Group 2 influenzaviruses. Thus the presently disclosed method of sequential vaccinationwith unique hemagglutinins enables the induction of broadly protectiveheterosubtypic antibodies against other conserved sites on influenzaviruses, such as on the stem of the H3 HA (Wang et al. 2010) and alsoother pathogens.

Recently, Wrammert et al (2011), reported very similar results ofmonoclonal antibodies (mAbs) generated from humans infected withpandemic H1N1 influenza. Thus 5 of the 15 mAbs against HA (30%)generated from 3 out of 4 people infected with pandemic H1N1 influenzawere against the HA-stem and neutralized a broad range of H1N1 influenzaviruses. Moreover Wrammert et al. (2011) observed that 4 of themonoclonal antibodies (two of which were a clonal pair) from 2 subjectsused IGHV1-69. They found that a total of 11 out of 15 monoclonalantibodies against the hemagglutinin of nH1N1 neutralized the pandemicH1N1 influenza, of which 5 were against the hemagglutinin stem. Only twoantibodies specifically neutralized the pandemic H1N1 and did notneutralize seasonal H1N1 influenza viruses. Thus 82% of the 15monoclonal antibodies that neutralized pandemic influenza alsoneutralized various seasonal H1N1 influenza viruses. Wrammert et al(2011) also found that there were a high number of somatic mutations inthe IGHV gene of the monoclonal antibodies and concluded, mostantibodies against the pandemic influenza were cross-reactive and weregenerated from memory B cells induced by seasonal influenza viruses orvaccines and said if this “is true it will be important to characterizethe efficacy of the pandemic H1N1 vaccine to induce a similarlycross-protective response.” The news of their findings provokedwide-spread public and scientific interest in its novel implications forthe prospect of a “universal influenza vaccine”. Thus Settembre et al.(2011) concluded “It now appears that sequential infection by virusstrains that share a conserved neutralizing epitope on a background ofsignificant antigenic change may promote the production of antibodiesagainst that conserved epitope. Such sequential exposures should promotegreater breadth of immunity”. Recently there has been a report of theisolation of a human monoclonal antibody from a human vaccinated withthe seasonal influenza vaccine that cross-protected against most Group 2hemagglutinin subtype viruses, including H3N2 and H7N7 viruses (Ekiertet al, 2011). This further supports that there are conserved sites onthe hemagglutinin stem in Group 2 viruses (Wang et al, 2010) and isfurther evidence that our invention of vaccination with successiveimmunizations of “unfamiliar”, unique hemagglutinins from Group 2, likeavian influenza viruses from Group 2, can result in circulatingcross-protective antibodies against Group 2 viruses.

Additionally, the present results show that the standardmicroneutralization tests and the hemagglutination inhibition assay areinadequate to monitor the degree of protection in serum induced by thisvaccination strategy.

To conclude, the present inventor has demonstrated that humans canrespond to infection or vaccination with an influenza with a uniquehemagglutinin by generating PB and memory B cells makingcross-protective antibodies against HA, including those using IGHV1-69which encodes in the germline, amino acid residues that make keycontacts with a conserved epitope on HA of many subtypes of influenza(Ekiert et al. 2009; Sui et al. 2009). Thus, when care is taken to avoidactivation of strain-specific memory B cells, most subjects are able tomake such cross-reactive or heterosubtypic protective antibodies,providing a broad-spectrum influenza vaccine.

The present disclosure shows that the nH1N1 vaccine, prepared using thetemplate used for the preparation of conventional seasonal influenzavaccines every year and given safely to millions of humans, can induceunprecedented levels of protective heterosubtypic, antibodies againstthe hemagglutinin stem.

Further, there is among those skilled in the art, much knowledge of theproduction of gp140 protein or viral vectors expressing gp160 immunogensthat are capable of inducing neutralizing antibodies against HIV-1,albeit against a restricted number of isolates of HIV-1 (KarlssonHedstam et al, 2008, Kwong and Wilson, 2009 and Rerks-Ngarm et al,2009). The present use of these gp140 proteins or DNA or viral vectorsexpressing gp160 in HIV-1 vaccines involve repeated immunization withgp140 or gp160 from the same or a similar isolate of HIV-1. Therationale is that broadly neutralizing antibodies are very rare and havemany somatic mutations and have unusual structures that prolongedimmunization with the same antigen or similar antigens (for example witha protein gp140 and a DNA or viral vector expressing a similar gp160) isnecessary (Karlsson Hedstam et al, 2008, Kwong and Wilson, 2009 andRerks-Ngarm et al, 2009). In contrast, the present disclosure teachesthat a dominant broadly neutralizing antibody response against influenzavirus can be stimulated in humans with vaccination with a uniqueantigen, in this case HA. This in striking contrast to the weak and lowheterosubtypic antibody response to repeated vaccination with driftedversions of seasonal influenza (Corti et al. 2010 and Wrammert et al.2011). Thus, existing HIV-1 gp140 or gp160 antigens that aresubstantially antigenically different and are from different Clades ofHIV-1 (for example from Clade A and Clade B) can be re-deployed asunique antigens used according to the presently disclosed methodology,to induce broadly neutralizing antibodies against HIV-1.

Methods

These studies were approved by the Research Ethics Boards of theUniversity of British Columbia and the University of Toronto. Blood wascollected from convalescent patients with laboratory-confirmed nH1N1infections or from subjects vaccinated with the adjuvanted nH1N1influenza vaccine (Arepanrix™). Peripheral blood monocytes (PBMC) andplasma samples were prepared and frozen. RT-PCR and cloning of cDNAencoding the variable regions of antibodies (McLean et al. 2005) wasperformed with three cellular sources, in two cases individual PBpurified by FACS, either randomly chosen or purified by multicolour FACSas binding to fluorescently labelled nH1 HA, and in the third, singleclones expanded from FACS-purified single memory B cells that bound tofluorescent nH1 HA as described in PCT/CA2006/001074. PB studied werecollected 7-10 days after exposure to nH1N1 antigens and memory B cells2-8 weeks. IgG1 monoclonal antibodies were transiently expressed andpurified as before (McLean et al. 2005), and binding to purifiedtrimeric recombinant snH1 HA was determined through an ELISA. nH1N1microneutralization assays were performed as outlined by the WHO Manualon Animal Influenza Diagnosis and Surveillance (see world wide web atwpro.who.int/internet/resources.ashx/CSR/Publications/manual+on+animal+ai+diagnosis+and+surveillance.pdf).Briefly, the monoclonal antibody was subjected to 2-fold serialdilutions in a microtitre plate beginning at 1:2 and an equal volume ofpdmH1N1 virus containing 100 TCID50 were added to each dilution andincubated for 2 h at 37° C. The mixtures were added to respective wellsof a microtitre plate containing monolayers of MDCK cells in serum-freeMegavir medium containing TCPK-treated trypsin and incubated for 3 hafter which the medium was replaced by fresh Megavir medium containingTCPK-treated trypsin. The monolayers were monitored on days 3, 4 and 5for the development of cytopathic effect (CPE). The reciprocal of thehighest dilution of the antibody that inhibited the development of viralCPE was designated as the titre. A modified form of the assay consistedof eliminating the step of omitting the removal of the virus-antibodymixtures after 3 h of incubation and allowing the mAb and the virus toremain in the medium for the duration of the assay. The standard WHOneutralization assay was performed in two experienced public healthlaboratories and did not reveal neutralization by anti-HA stem mAbs. Wereasoned that in the standard assay the mixture of the viruses and mAbwill only be in contact with the host cells for 3 hr and, if the mAbtargets the HA stem rather than the head, the viruses will successfullyattach to the host cells. After the virus and the mAb are washed away,some of the mAb will dissociate from viruses that are not endocytosedbut attached to the cells. When the virus is finally endocytosed theremaybe insufficient mAb bound to the virus to inhibit fusion. Thus thevirus will replicate and can infect other cells, unimpeded by the mAb asit is no longer present. In the modified assay, when the anti-HA stemmAbs were left for the full 3-4 days of the assay, neutralization wasobserved. The standard WHO assay was designed to detect neutralizingantibodies in serum. Therefore the mixture of virus and titrations ofsera was washed away after 3 hr incubation with the host cells becauseserum contained trypsin inhibitors that blocked the trypsin that was anessential component of the assay. The action of the trypsin wasnecessary to cleave the hemagglutinin on the virus and render itinfectious to the host cells in order to see a cytopathic effect. Thisformat worked well with seasonal influenza where the neutralizingantibodies were directed at the HA head and blocked viral attachment.

H5N1 Plaque Reduction Assay:

˜20,000 plaque forming units (PFU)/ml of influenza virusesA/Goose/Ger/R1400/07 (H5N1) and A/Ck/Ger/R28/03 (H7N7) were incubatedwith the indicated mAbs at three different concentrations (40 μg/mL, 20μg/mL, and 10 μg/mL) at 37° C. for 1 h. 50 μl of a 1:10 dilution of thevirus-antibody mixtures (containing ˜100 PFU) were transferred intriplicate onto MDCK cell monolayers in 96 well plates and incubated for1 h at 37° C. Cells were then washed and overlaid with 1.5% CMC. 30 hlater, plaques were visualized by immunostaining using a mousemonoclonal anti-NP antibody (F26NP9-2-1) (Weingartl et al 2010). Plaqueswere counted and % neutralization was calculated by setting theinfection without mAb as 0% neutralization. Data shown are the mean oftriplicate measurements with SD.

Binding to Cell-Expressed H5 HA:

Recombinant adenoviruses expressing influenza A HA (AdHA) from A/HongKong/156/97 (H5N1) (Hoelscher, M. A., et al, 2008) were provided by Dr.Suryaprakash Sambhara (Centres for Disease Control and Prevention,Atlanta, Ga.). The AdHA proprotein convertase (AdHA-PC) containing theHA multi-basic cleavage site was utilized. The Adenovirus empty(AdEmpty) was used as a negative control (Viraquest Inc). To promote HAstability, NH₄Cl (10 mM) was added to the culture medium during AdHAinfection. Monolayers of A549 cells in 96-well plates were infected withAdHA at a MOI of 500 and incubated for 40 h prior to fixation with 4%formaldehyde. Then, cells were incubated with the indicatedconcentrations of mAb and treated with FITC-labelled anti-humanimmunoglobulin antibodies. Nuclei were counterstained with Hoechst.Images were recorded and analyzed/counted using the Cellomics instrument(Thermo Scientific). Data shown are the average of duplicatemeasurements from a representative experiment.

Assay for Inhibition of HA-Mediated Fusion:

A549 cells were seeded and infected with AdHA as described in the methodfor the binding assay. At 40 h post infection, cells were washed withPBS, and incubated with the indicated antibodies (20 μg/ml) for 30 minat 37° C. Then, cells were washed again and treated with fusion buffer(10 mM HEPES, 10 mM MES, pH 5) for 5 min at room temperature. Media wasreplaced with normal cell culture media and cells were incubated at 37°C. for a 5 h period to allow for syncytia formation. To monitor syncytiaformation, cells were labelled with 10 μM Cell Tracker Green CMFDA(Molecular Probes) for 30 min at 37° C., followed by further incubationfor 30 min in fresh media before samples were fixed with 4%formaldehyde. Nuclei were counterstained with Hoechst dye (1 μg/ml).Images were analyzed using the Cellomics system.

Therapeutic and Prophylactic Testing of Antibodies Against Fatal pdmH1N1and H5N1 Pneumonia in Mice:

The human clinical isolate of pandemic H1N1 influenza,A/Halifax/210/2009 or the H5N1 vaccine strain, A/Hong Kong/213/2003 onthe PR8/34 backbone were used to induce fatal viral pneumonia in CD-1mice or BALB/c mice respectively. Groups of 5 CD-1 or BALB/c mice (19-21Gm, females) were infected under halothane anaesthesia (3.5% in O₂) with50 μL of 2×10⁵ PFU of either H5N1 or pmdH1N1 virus in PBS. Mice wereeither treated with an intraperitoneal injection of 0.5 ml of purifiedmonoclonal antibodies 1 day before or 1-2 days after infection tomonitor protective or therapeutic effects. Controls were treated withonly PBS. Survival and weight-loss was monitored for 12-14 days toassess the effects against fatal pandemic H1N1 or H5N1 infections inmice. Similarly, mice were either treated with an intraperitonealinjection of 0.4 ml of human plasma 1 day before or 0.4 ml of humanplasma for each of 3, 2 or 1 days before infection with H5N1 to monitorprotective effects.

Statistical Analysis: Statistical analyses were carried out in R(Version 2.9.1., The R Foundation for Statistical Computing, see worldwide web at r-project.org) and Sigmaplot 11.2. Continuous variables werecompared using the Mann-Whitney rank sum. For estimation of theprobability of V-gene usage we used the binomial distribution.

While the present disclosure has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the disclosure is not limited to the disclosed examples.To the contrary, the disclosure is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Human monoclonal antibodies binding recombinant hemagglutininfrom nH1N1 mAb IGHV D J κ/λ IGK/LV J Mutations (A) mAb generated fromplasmablasts I3-15 1-69*01 3-10 6 κ 1-39*01 2 18   I4-112^(#)  1-2*026-19 3 λ  2-8*01 2 5   I4-115^(#) 3-20*01 3-10 4 κ 3-11*01 1 30  I4-128^(#) 1-69*01 5-5  5 κ 3-11*01 1 13   I4-109^(#) 3-33*01 4-17 4 λ1-40*01 2 16 I5-24 1-69*02 3-22 4 κ  3-20*001 2 29 I5-52 1-18*01 3-16 6κ 2-30*01 1 11 I5-69 1-69*02 5-24 4 κ 3-11*01 5 8 I5-7  1-69*12 3-10 3 κ3-15*01 1 29 V2-1  3-30*18 1-26 6 κ 3-20*01 1 15 V2-2  4-39*01 6-13 5 λ1-40*01 1 33 V2-3  4-39*01 6-13 5 λ 1-40*01 1 33 V2-4  4-39*01 6-13 5 λ1-40*01 1 31 V2-11  4-39*01 6-13 5 λ 1-40*01 1 21 V2-36  4-39*01 6-13 5λ 1-40*01 1 33 V2-38  4-39*01 6-13 5 λ 1-40*01 1 34 V2-7  4-39*01 6-13 5λ 1-40*01 1 21 V2-12  4-39*01 3-22 4 κ  4-1*01 3 34 V4-1  1-69*06 5-12 4κ 3-20*01 2 29 V4-9  1-69*02 7-27 5 κ 3-15*01 1 34 V4-12  3-33*02 5-24 4λ  3-1*01 3 31 V4-17^(H) 4-39*01 3-3  4 λ 1-47*02 2 19 V4-18  1-69*065-12 4 κ 3-20*01 2 23 V4-23  1-69*02 7-27 6 κ 1-39*01 1 34 V4-29 1-69*01 5-5  6 λ 2-14*01 2 28 (B) mAb generated from memory B cells I4-1G8 2-70*11 2-8  6 λ  3-1*01 1 5  I4-1C4 1-69*09 4-23 5 κ 3-11*01 123  I4-1F7 3-66*01 1-26 2 κ 3-15*01 3 0  I8-1B6 1-69*01 3-3  6 λ 2-14*011 22 I14-2B7 1-18*01 2-8  6 κ 2-30*01 2 3 I14-1F8 5-51*01 5-12 6 κ3-11*01 5 10 I14-2B6 4-59*01 2-8  3 κ 1-39*01 1 19 I14-2C5 3-30*03 6-6 4 κ  1-6*01 2 22 I14-1D9 1-69*04 2-15 4 κ 3-15*01 2 10 V3-1B9 3-11*013-3  3 κ 1-12*01 4 10 V3-1C6 1-69*01 1-1  6 λ 7-43*01 3 18 V3-1E83-33*01 1-26 4 λ 3-25*03 3 11  V3-2C10 1-69*01 5-12 5 κ 1-39*01 4 21V3-2C2 1-69*06 3-9  4 κ 1-39*01 4 9 V3-2C3 1-69*01 3-10 5 κ  1-5*03 2 6 V3-2F10 1-69*06 3-22 4 κ  1-5*03 1 17 V3-2G6 1-69*01 3-16 4 κ 3-20*01 218 V3-3B3 1-69*01 3-10 3 λ 2-11*01 1 6 V3-3B6 1-69*06 2-2  6 κ 3-20*01 119 V3-2E2 1-69*01 3-10 4 κ 1-16*02 4 14  V3-3C11 1-69*01 5-12 4 λ2-14*01 1 9  V3-1G10 1-69*06 1-14 5 κ 3-15*01 2 25 V3-3D2 1-69*01 1-14 6κ  1-16*−01 4 21 mAbs are named systematically, with the initial letterand number indicating the subject from which it had been generated, withI or V indicating that they were generated from a subject infected (I)or vaccinated (V) with the nH1N1 virus. Mutations listed are in IGHV.All mAbs bound by ELISA to purified recombinant ectodomain of HA ofnH1N1 and to the nH1N1 vaccine, and all but V4-17 (*) also bound to theseasonal 2009/2010 vaccine and to H5 HA (A/Vietnam/1203/2004, Clade l).mAbs in section (A) were generated from PB, with # indicating thosegenerated from randomly chosen PB that were not sorted for their abilityto bind to HA from nH1N1; other mAbs in section (A) were generated fromPB purified by their binding to to HA from nH1N1. mAbs in section (B)were generated from memory B cells purified by their binding to to HAfrom nH1N1.

TABLE 2 Sequences: The CDRs as defined by IGMT are underlined. Alsogiven (but not underlined) are amino acids in the framework close to theCDRs that are mutated. The latter residues may be replaced in the frameworkwhen these CDR's are transplanted into an immunoglobulin framework torecreate the binding site of these antibodies. 1 V2-36 Seq 1HELRLHESGPGLVKPSGTLSLTCTVSGGSISGGSHYWAWIRQSPGKGLEWIG SIYYSGSTYDSPSLKSRLSMSVDKSKNQFHLTLRSVTAADTAVYFCAKHESDSSSWHTGWNWFDP (SEQ ID NO: 8) Seq1LQSVLTQPSSVSGAPGQRVTISCTGSYSNIGTGFDVHWYQHLPGKAPKLLIFGNN NRPSGVPDRFSGSKSGTSASLAITGLQPEDEGDYYCQSFDSSLSGSNV (SEQ ID NO: 7)Seq1cdrh1 GGSISGGSHY WA (SEQ ID NO: 4 - underlined, SEQ ID NO: 81)Seq1cdrh2 IYYSGST YDS (SEQ ID NO: 5 - underlined, SEQ ID NO: 102)Seq1cdrh3 AKHESDSSSWHTGWNWFDP (SEQ ID NO: 6)Seq1cdrl1 YSNIGTGFD (SEQ ID NO: 1) Seq1cdrl2 GNN (SEQ ID NO: 2)Seq1cdrl3 QSFDSSLSGSNV (SEQ ID NO: 3) 2 V2-7 Seq2hQLQLQESGPGLVKTSETLSLTCTVSGGSIRGGTNYWAWIRQPPGKGPEWLGSVYYSGSTYDNPSLKSRVSIYVDTSKNKFSLRLRSVTAADTAIYYCARHESDSSSWHTGWNWFDPWGQGTLVTVSSAS (SEQ ID NO: 16) Seq2lQSMLTQPPSVSGAPGQRVTISCTGSSTNIGAGLAVHWYQHLPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTTASLAITGLQADDEADYYCQSFDGSLSGSNV FGTGTKVTVLTAA(SEQ ID NO: 15)Seqcdrh1 GGSIRGGTNY WA (SEQ ID NO: 12 - underlined, SEQ ID NO: 82)Seqcdrh2 VYYSGSTYD (SEQ ID NO: 13 - underlined, SEQ ID NO: 83)Seqcdrh3 ARHESDSSSWHTGWNWFDP (SEQ ID NO: 14)Seqcdrl1 STNIGAGLA (SEQ ID NO: 9) Seqcdrl2 GNT (SEQ ID NO: 10)Seqcdrl3 QSFDGSLSGSNV (SEQ ID NO: 11) 3 V3-2G6 Seq3hQSQVQLEQSGAEVKRPGSSVKVSCQTSGGTFSSFAFSWVRQAPGQGLEWVGG IIGMFGTTSYAQKFQGRVTISADESTSTAYMELSSLRSDDTAIYYCARGKKYYHDTLDY (SEQ ID NO: 24) Seq3lEIVLTQSPGTLSLSPGERATLSCRASQIVSSSQLAWYQHKPGQAPRLLIYAAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTSHA (SEQ ID NO: 23)Seq3cdrh1 GGTFSSFA F (SEQ ID NO: 20 - underlined, SEQ ID NO: 84)Seq3cdrh2 IIGMFGTT S (SEQ ID NO: 21 - underlined, SEQ ID NO: 85)Seq3cdrh3 ARGKKYYHDTLDY (SEQ ID NO: 22)Seq3cdrl1 QIVSSSQ (SEQ ID NO: 17) Seq3cdrl2 AAS (SEQ ID NO: 18)Seq3cdrl3 QQYGTSHA (SEQ ID NO: 19) 4. I8-1B6 Seq4hQAQLEQSGAEVRRPGSSVKVACKTSGGIFSNFAVSWVRQAPGQGLEWMGGILSIFRTTNYAQKFQGRVTITADESTSTAYMELNSLRSDDTAVYYCARSITNLYYYYMDV (SEQ ID NO: 32) Seq4lQSALTQPASVSGSPGQSITVSCTGTNSDVGTYNYVSWFQQHPGEAPKVIIFDVS HRPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYCSSYTTSNTRV (SEQ ID NO: 31)Seq4cdrh1 GGIFSNFA V (SEQ ID NO: 28 - underlined, SEQ ID NO: 86)Seq4cdrh2 ILSIFRTT (SEQ ID NO: 29)Seq4cdrh3 ARSITNLYYYYMDV (SEQ ID NO: 30)Seq4cdrl1 NSDVGTYNY (SEQ ID NO: 25)Seq4cdrl2 DVS H (SEQ ID NO: 26 - underlined, SEQ ID NO: 87)Seq4cdrl3 SSYTTSNTRV (SEQ ID NO: 27) 5 V3-3D2 Seq5hQVQLVQSGAEVKKPGSSVKVSCKAPGVIFNAYAMSWVRQAPGQGLEWMGGITGVFHTATYAPKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARGPKYYHSYMDV (SEQ ID NO: 40) Seq5lDIQMTQSPSSLSASVGDRVTITCRASQDISNYVAWFQQKPGKTPKSLMYATS KLQNGVPSRFSGSGSGTDFTLTISSLQSEDFATYYCQQYSRYPPT (SEQ ID NO: 39)Seq5cdrh1 P GVIFNAYA M (SEQ ID NO: 36 - underlined, SEQ ID NO: 88)Seq5cdrh2 ITGVFHTA T (SEQ ID NO: 37 - underlined, SEQ ID NO: 89)Seq5cdrh3 ARGPKYYHSYMDV (SEQ ID NO: 38)Seq5cdrl1 QDISNY V (SEQ ID NO: 33 - underlined, SEQ ID NO: 90)Seq5cdrl2 MY ATS K (SEQ ID NO: 34 - underlined, SEQ ID NO: 91)Seq5cdrl3 QQYSRYPPT (SEQ ID NO: 35) 6 V3-1G10 Seq6hQVQLVQSGAEVKKPGSTVKVSCEASGVTFNHYTVSWVRQAPGQGLEWMGG IIPLFGTADYAQKFQDRVTITADRSTGTAYMELSSLRPEDTALYYCARSGTTKTRYNWFDP (SEQ ID NO: 48) Seq6lEIIMTQSPATLSLSPGERVTLSCRASQSVGTNLAWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSETEFTLSISSLQSEDFAVYYCQHYNNWPPYT (SEQ ID NO: 47)Seq6cdrh1 EAS GVTFNHYT V (SEQ ID NO: 44 - underlined, SEQ ID NO: 92)Seq6cdrh2 IIPLFGTA D (SEQ ID NO: 45 - underlined, SEQ ID NO: 93)Seq6cdrh3 ARSGTTKTRYNWFDP (SEQ ID NO: 46)Seq6cdrl1 QSVGTN (SEQ ID NO: 41)Seq6cdrl2 F GAS (SEQ ID NO: 42 - underlined, SEQ ID NO: 94)Seq6cdrl3 QHYNNWPPYT (SEQ ID NO: 43) 7 I5-24 Seq7hQFQLVQSGAEVRKPGSSVKVSCTASGGTFSRYTVNWVRQAPGQGLQWMGR FIPLLGMTNYAQRFQGRATITADKSTTTAFLELSSLTSEDTAVYFCARHDSSGYHPLDY (SEQ ID NO: 56) Seq7lEIVLTQSPGTLSLSPGERATLSCRASQSLSSGHLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYAVFLYT (SEQ ID NO: 55)Seq7cdrh1 TAS GGTFSRYT VN (SEQ ID NO: 52 - underlined, SEQ ID NO: 95)Seq7cdrh2 FIPLLGMT (SEQ ID NO: 53)Seq7cdrh3 ARHDSSGYHPLDY (SEQ ID NO: 54)Seq7cdrl1 QSLSSGH (SEQ ID NO: 49) Seq7cdrl2 GAS (SEQ ID NO: 50)Seq7cdrl3 QQYAVFLYT (SEQ ID NO: 51) 8 I4-128 Seq8hQVQLVQSGAEVKKPGSSVMVSCKASGGTFSTYGVSWVRQAPGQGLEWVGG IIPIFGTAKYAQKFQGRVTITADESSTTAYMELSRLRSEDTAVYYCARPNTYGYILPVY (SEQ ID NO: 64) Seq8lDIQMTQSPSTLSASVGDRVTIGCRASQTISTYLAWYQQVPGKAPKLLIYMASTLESGVPSRFSGSGSGTEFTLTISSLQPGDFATYYCQHYNTYSST (SEQ ID NO: 63)Seq8cdrh1 GGTFSTYG V (SEQ ID NO: 60 - underlined, SEQ ID NO: 96)Seq8cdrh IIPIFGTA K (SEQ ID NO: 61 - underlined, SEQ ID NO: 97)Seq8cdrh3 ARPNTYGYILPVY (SEQ ID NO: 62) Seq8cdrl1 QTISTY (SEQ ID NO: 57)Seq8cdrl2 MAS T (SEQ ID NO: 58 - underlined, SEQ ID NO: 98)Seq8cdrl3 QHYNTYSST (SEQ ID NO: 59) 9 V4-17 Seq9hQLQLQESGPGLVKPSETLSLTCTVSGGSITRNSYFWGWIRQPPGKGLEWIG SMYYDGTTYHNPSLKSRLTLSADTSKNQFSVRLSSVTAADTAVYYCARHHVTELRVLEWLPKSDY (SEQ ID NO: 72) Seq9lQSVLTQPPSASGTPGQRVTISCSGSSSNIGTYYVHWYQHLPGTAPKLLIYDNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYHCAAWDDSLSGVV (SEQ ID NO: 71)Seq9cdrh1 GGSITRNSYF (SEQ ID NO: 68)Seq9cdrh2 MYYDGTT YH (SEQ ID NO: 69 - underlined, SEQ ID NO: 99)Seq9cdrh3 ARHHVTELRVLEWLPKSDY (SEQ ID NO: 70)Seq9cdrl1 SSNIGTYY VH (SEQ ID NO: 65 - underlined, SEQ ID NO: 100)Seq9cdrl2 DNN (SEQ ID NO: 66) Seq9cdrl3 AAWDDSLSGVV (SEQ ID NO: 67)10 V3-2C3 Seq10h QVQLVQSGAEVKKPGSSVKVSCKASGGTFNNYAVSWVRQAPGQGLEWMGGIIPIFGTANYAHKFQGRVTITVDESTSTAYMELSSLRSEDTAMYYCARVCSFYGSGSYYNVFCY (SEQ ID NO: 80) Seq10lDIQMTQSPSTLSASAGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYNSYSQTFGQGTKVEIKRTAAA (SEQ ID NO: 79)Seq10cdrh1 GGTFNNYA V (SEQ ID NO: 76 - underlined, SEQ ID NO: 101)Seq10cdrh2 IIPIFGTA (SEQ ID NO: 77)Seq10cdrh3 ARVCSFYGSGSYYNVFCY (SEQ ID NO: 78)Seq10cdrl1 QSISSW (SEQ ID NO: 73) Seq10cdrl2 KAS (SEQ ID NO: 74)Seq10cdrl3 QHYNSYSQT (SEQ ID NO: 75)

TABLE 3 Expected versus observed IGHV1-69 usage of mAbs binding to snHAper subject # of anti-snHA mAbs *Probability of Subject Total UsingIGHV1-69 observed result I3 1 1 0.04 I4 8 2 0.03 I5 4 3 0.0002 I8 1 10.04 I14 5 1 0.2 V3 14 12 1.E-15 V4 7 5 2E-6  V2 9 0 0.7 *based on theaverage frequency of usage of IGHV1-69 in human antibodies of 4%. Notethat all but 2 of the 8 subjects (V2 and I14) had a greater thanexpected frequency of IGHV1-69-using mAbs. The total proportion of mAbsusing IGHV1-69 was 52% (95% confidence intervals of 38-66%). If it ishypothesized that in the response to nH1N1, IGHV1-69 is used in 52% ofmAbs, the probability that the results obtained with subject V2 occurredby chance was only 0.002, whereas for I14 the chance was 0.16 (i.e. waslikely).

REFERENCES

-   Allen, C. D., Okada, T., Tang, H. L. & Cyster, J. G. Imaging of    germinal center selection events during affinity maturation. Science    315, 528-531 (2007).-   Babcook, J. S., Leslie, K. B., Olsen, O. A., Salmon, R. A. &    Schrader, J. W. A novel strategy for generating monoclonal    antibodies from single, isolated lymphocytes producing antibodies of    defined specificities. Proceedings of the National Academy of    Sciences of the United States of America 93, 7843-7848 (1996).-   Bardiya, N. & Bae, J. H. Influenza vaccines: recent advances in    production technologies. Appl Microbiol Biotechnol 67, 299-305,    doi:10.1007/s00253-004-1874-1 (2005).-   Barington, T., Heilmann, C. & Andersen, V. Quantitation of    antibody-secreting cells in the blood after vaccination with    Haemophilus influenzae type b conjugate vaccine [published erratum    appears in Scand J Immunol 1990 July; 32(1):59]. Scand J Immunol 31,    515-522 (1990).-   Burioni, R. et al. Monoclonal antibodies isolated from human B cells    neutralize a broad range of H1 subtype influenza A viruses including    swine-origin Influenza virus (S-OIV). Virology 399, 144-152.-   Chen, G. L. & Subbarao, K. Attacking the flu: neutralizing    antibodies may lead to ‘universal’ vaccine. Nat Med 15, 1251-1252    (2009).-   Corti, D. et al. Heterosubtypic neutralizing antibodies are produced    by individuals immunized with a seasonal influenza vaccine Journal    of Clinical Investigation 120, 1663-1673 (2010).-   de Wildt, R. M., Hoet, R. M., van Venrooij, W. J., Tomlinson, I. M.    & Winter, G. Analysis of heavy and light chain pairings indicates    that receptor editing shapes the human antibody repertoire. Journal    of Molecular Biology 285, 895-901 (1999).-   Ding, N., Wu, N., Xu, Q., Chen, K. & Zhang, C. Molecular evolution    of novel swine-origin A/H1N1 influenza viruses among and before    human. Virus Genes 39, 293-300 (2009).-   Doherty, P. C. & Kelso, A. Toward a broadly protective influenza    vaccine. J Clin Invest 118, 3273-3275 (2008).-   Eisenlohr, L. C., Gerhard, W. & Hackett, C. J. Role of    receptor-binding activity of the viral hemagglutinin molecule in the    presentation of influenza virus antigens to helper T cells. Journal    of Virology 61, 1375-1383 (1987).-   Ekiert, D. C. et al. Antibody recognition of a highly conserved    influenza virus epitope. Science 324, 246-251 (2009).-   Ekiert, D. C., et al. A Highly Conserved Neutralizing Epitope on    Group 2 Influenza A Viruses. Science. (2011) July 7 Epub ahead of    print-   Garcia, J. M. et al. Heterosubtype neutralizing responses to    influenza A (H5N1) viruses are mediated by antibodies to virus    haemagglutinin. PLoS One 4, e7918, (2009).-   Garten, R. J., et al. Antigenic and genetic characteristics of    swine-origin 2009 A(H1N1) influenza viruses circulating in humans.    Science 325, 197-201 (2009).-   Gioia, C. et al. Cross-subtype immunity against avian influenza in    persons recently vaccinated for influenza. Emerging Infectious    Diseases 14, 121-128 (2008).-   Hancock, K. et al. Cross-Reactive Antibody Responses to the 2009    Pandemic H1N1 Influenza Virus. N Engl J Med (2009).-   Hellmann, C., Henrichsen, J. & Pedersen, F. K. Vaccination-induced    circulation of human B cells secreting type-specific antibodies    against pneumococcal polysaccharides. Scand J Immunol 25, 61-67    (1987).-   Hoelscher, M. A., et al. A Broadly Protective Vaccine against    Globally Dispersed Clade 1 and Clade 2 H5N1 Influenza Viruses. The    Journal of Infectious Diseases 197, 1185-1188 (2008).-   Itoh, Y. et al. In vitro and in vivo characterization of new    swine-origin H1N1 influenza viruses. Nature 460, 1021-1025 (2009).-   Karlsson Hedestam, G. B., et al. The challenges of eliciting    neutralizing antibodies to HIV-1 and to influenza virus. Nat Rev    Micro 6, 143-155 (2008).-   Kashyap, A. K. et al. Combinatorial antibody libraries from    survivors of the Turkish H5N1 avian influenza outbreak reveal virus    neutralization strategies. Proc Natl Acad Sci USA 105, 5986-5991    (2008).-   Klenk, H. D., Rott, R., Orlich, M. & Bloedorn, J. Activation of    influenza A viruses by trypsin treatment. Virology 68, 426-439    (1975).-   Kropff, B., Landini, M. P. & Mach, M. An ELISA using recombinant    proteins for the detection of neutralizing antibodies against human    cytomegalovirus. J Med Virol 39, 187-195 (1993).-   Lynch, G. W., Selleck, P. & Sullivan, J. S. Acquired heterosubtypic    antibodies in human immunity for avian H5N1 influenza. 3, 205-209    (2009).-   McLean, G. R. et al. Recognition of human cytomegalovirus by human    primary immunoglobulins identifies an innate foundation to an    adaptive immune response. J Immunol 174, 4768-4778 (2005).-   McLean, G. R., Cho, C. W. & Schrader, J. W. Autoreactivity of    primary human immunoglobulins ancestral to hypermutated human    antibodies that neutralize HCMV. Mol Immunol 43, 2012-2022 (2006).-   Nossal, G. J. V. & Lewis, H. Variation In Accessible Cell Surface    Immunoglobulin Among Antibody-Forming Cells. The Journal of    Experimental Medicine 135, 1416-1422 (1972).-   Ohlin, M., Sundqvist, V. A., Mach, M., Wahren, B. &    Borrebaeck, C. A. Fine specificity of the human immune response to    the major neutralization epitopes expressed on cytomegalovirus    gp58/116 (gB), as determined with human monoclonal antibodies. J    Virol 67, 703-710 (1993).-   Odendahl, M. et al. Generation of migratory antigen-specific plasma    blasts and mobilization of resident plasma cells in a secondary    immune response. Blood 105, 1614-1621 (2005).-   Okuno, Y., Isegawa, Y., Sasao, F. & Ueda, S. A common neutralizing    epitope conserved between the hemagglutinins of influenza A virus H1    and H2 strains. J Virol 67, 2552-2558 (1993).-   Paus, D. et al. Antigen recognition strength regulates the choice    between extrafollicular plasma cell and germinal center B cell    differentiation. The Journal of Experimental Medicine 203, 1081-1091    (2006).-   Pinna, D., Corti, D., Jarrossay, D., Sallusto, F. & Lanzavecchia, A.    Clonal dissection of the human memory B-cell repertoire following    infection and vaccination. Eur J Immunol 39, 1260-1270,    doi:10.1002/eji.200839129 (2009).-   Rerks-Ngarm, S., et al. Vaccination with ALVAC and AIDSVAX to    prevent HIV-1 infection in Thailand. The New England Journal of    Medicine 361, 2209-2220 (2009).-   Russell, S. M. & Liew, F. Y. T cells primed by influenza virion    internal components can cooperate in the antibody response to    haemagglutinin. Nature 280, 147-148 (1979).-   Sagawa, H., Ohshima, A., Kato, I., Okuno, Y. & Isegawa, Y. The    immunological activity of a deletion mutant of influenza virus    haemagglutinin lacking the globular region. J Gen Virol 77,    1483-1487 (1996).-   Sasso, E. H., Johnson, T. & Kipps, T. J. Expression of the    immunoglobulin VH gene 51p1 is proportional to its germline gene    copy number. Journal of Clinical Investigation 97, 2074-2080 (1996).-   Settembre, E. C., Dormitzer, P. R. & Rappuoli, R. Learning from the    2009 H1N1 pandemic: prospects for more broadly effective influenza    vaccines. J Mol Cell Biol 3, 144-146. (2011).-   Schwickert, T. A., et al. A dynamic T cell limited checkpoint    regulates affinity-dependent B cell entry into the germinal center    The Journal of Experimental Medicine. 208, 1243-1252 (2011)-   Smith, K. G., Light, A., Nossal, G. J. & Tarlinton, D. M. The extent    of affinity maturation differs between the memory and    antibody-forming cell compartments in the primary immune response.    Embo J 16, 2996-3006 (1997).-   Steel, J. et al. Influenza Virus Vaccine Based on the Conserved    Hemagglutinin Stalk Domain. mBio1, e00018-00010 (2010).-   Sui, J. et al. Structural and functional bases for broad-spectrum    neutralization of avian and human influenza A viruses. Nat Struct    Mol Biol 16, 265-273 (2009).-   Tangye, S. G. & Tarlinton, D. M. Memory B cells: effectors of    long-lived immune responses. Eur J Immunol 39, 2065-2075 (2009).-   Thomson, C. A. et al. Germline V-genes sculpt the binding site of a    family of antibodies neutralizing human cytomegalovirus. Embo J    (2008).-   Throsby, M. et al. Heterosubtypic neutralizing monoclonal antibodies    cross-protective against H5N1 and H1N1 recovered from human IgM+    memory B cells. PLoS One 3, e3942 (2008).-   Victora, G. D., et al. Germinal center dynamics revealed by    multiphoton microscopy with a photoactivatable fluorescent reporter.    Cell 143, 592-605 (2010)-   Wagner, B. et al. A continuous sequence of more than 70 amino acids    is essential for antibody binding to the dominant antigenic site of    glycoprotein gp58 of human cytomegalovirus. Journal of Virology 66,    5290-5297 (1992).-   Wang, T. T. et al. Broadly protective monoclonal antibodies against    H3 influenza viruses following sequential immunization with    different hemagglutinins. PLoS Pathog 6, e1000796. (2010).-   Wei, C.-J., et al. Cross-neutralization of 1918 and 2009 influenza    viruses: role of glycans in viral evolution and vaccine design.    Science Translational Medicine 2, 24ra21 (2010)a-   Wei, C.-J., at al. Induction of broadly neutralizing H1N1 influenza    antibodies by vaccination. Science (New York, N.Y.) 329, 1060-1064    (2010)b.-   Weingartl, H. M., et al. Genetic and Pathobiologic Characterization    of Pandemic H1N1 2009 Influenza Viruses from a Naturally Infected    Swine Herd. J. Virol. 84, 2245-2256 (2010).-   Wen, L., et al. Limiting dilution assay for human B cells based on    their activation by mutant EL4 thymoma cells: total and antimalaria    responder B cell frequencies. Eur J Immunol 17, 887-892 (1987).-   Wiley, D. C. & Skehel, J. J. The structure and function of the    hemagglutinin membrane glycoprotein of influenza virus. Annu Rev    Biochem 56, 365-394 (1987).-   Wrammert, J. et al. Rapid cloning of high-affinity human monoclonal    antibodies against influenza virus. Nature 453, 667-671 (2008).-   Wrammert, J., at al. Broadly cross-reactive antibodies dominate the    human B cell response against 2009 pandemic H1N1 influenza virus    infection. The Journal of Experimental Medicine 208, 181-193 (2011)-   Xu, R. et al. Structural Basis of Preexisting Immunity to the 2009    H1N1 Pandemic Influenza Virus. Science 328, 357-360 (2010).-   Yen, H.-L. & Webster, R. G. Pandemic influenza as a current threat.    Current Topics in Microbiology and Immunology 333, 3-24 (2009).-   Yoshida, R., et al. Cross-protective potential of a novel monoclonal    antibody directed against antigenic site B of the hemagglutinin of    influenza A viruses. PLoS Pathog 5, e1000350 (2009)

The invention claimed is:
 1. A method for inducing an antibody responseagainst a Group 1 or Group 2 influenza virus in a human subjectcomprising: (a) administering a first hemagglutinin (HA) protein to thehuman subject; and (b) administering a second HA protein to the humansubject 3-52 weeks after (a); wherein the first and second HA proteinscomprise heads that are antigenically unrelated to each other and toseasonal influenza but comprise conserved antigenic sites in the stem;wherein the stem is a Group 1 stem if the antibody response is against aGroup 1 influenza virus or the stem is a Group 2 stem if the antibodyresponse is against a Group 2 influenza virus; and wherein the Group 1or Group 2 influenza virus to which an antibody response is inducedcomprises a head that is antigenically unrelated to the first and secondHA proteins and to seasonal influenza but comprises the conservedantigenic sites in the stem of the first and second HA protein.
 2. Themethod of claim 1, wherein the first and/or second HA protein is part ofan attenuated influenza virus strain or inactivated influenza virus. 3.The method of claim 1, wherein the first and/or second HA protein isfrom a pandemic virus or a virus that infects a different host species.4. The method of claim 1, wherein the first and/or second HA protein isan artificial HA protein having mutations in the head that make the headantigenically unrelated to seasonal influenza or wherein the firstand/or second HA protein is a chimeric HA protein comprising theconserved stem coupled to an antigenically unrelated head.
 5. The methodof claim 1, further comprising administering an adjuvant with the firstand/or second HA protein.
 6. The method of claim 1, wherein the secondHA protein is administered at a different anatomical site from the sitewhere the first HA protein is administered.
 7. A kit comprising a firstand second hemagglutinin (HA) protein, wherein the first HA protein andthe second HA protein comprise a head that is antigenically unrelated toeach other and to seasonal influenza but comprise conserved antigenicsites in the stem, wherein the stem is a Group 1 stem if the antibodyresponse is against a Group 1 Influenza virus or the stem is a Group 2stem if the antibody response is against a Group 2 influenza virus. 8.The kit of claim 7, wherein the first and/or second HA protein is partof an attenuated influenza strain or inactivated influenza.
 9. The kitof claim 7, wherein the first and/or second HA protein is from apandemic influenza virus or an influenza virus that infects a differenthost.
 10. The kit of claim 7, wherein the first and/or second HA proteinis an artificial HA protein having mutations in the head that abolishits reaction with antibodies in the subject to seasonal influenza orwherein the first and/or second HA protein is a chimeric HA proteincomprising the conserved stem coupled to an antigenically unrelatedhead.
 11. The method of claim 1, further comprising: (c) administering athird HA protein 3-52 weeks after b), wherein the third HA proteincomprises a head that is substantially antigenically unrelated to thefirst or second HA protein or to seasonal influenza but comprises theconserved antigenic sites in the stem.
 12. The method of claim 1,further comprising administering one or more additional HA proteins as amixture or concurrently with the first and/or second HA protein; whereinthe one or more additional HA proteins comprise a head that isantigenically unrelated to the prior HA proteins or to seasonalinfluenza but comprises the conserved antigenic sites in the stem. 13.The method of claim 1, wherein the frequency of memory B cells to thefirst and/or second HA protein in the human subject is undetectable orless than 1 per million prior to administering the HA proteins.
 14. Themethod of claim 1, wherein the frequency of memory B cells to the firstand/or second HA protein in the human subject is less than 0.01% ofIgG-expressing memory B cells circulating in the blood of the humansubject prior to administering the HA proteins.
 15. The method of claim1, wherein the antibody response is induced against H5 influenza or H1influenza.
 16. The method of claim 15, wherein the antibody response isinduced against an H5N1 influenza virus or an H1N1 influenza virus. 17.The method of claim 1, wherein the stem is from a Group 1 influenzavirus.
 18. The method of claim 17, wherein the stem is from an H5influenza or an H1 influenza virus.
 19. The method of claim 1, whereinthe stem is from a Group 2 influenza virus.
 20. The method of claim 19,wherein the stem is from an H3 influenza virus.